Cryopreservation of plant cells

ABSTRACT

The present invention relates to methods for cryopreserving plant cells and to methods for recovering viable plant cells from long or short term cryopreservation. Plant cells to be cryopreserved can be grown in culture and pretreated with a solution containing an cryoprotective agent and, optionally, a stabilizer. Stabilizers are preferably membrane stabilizers such as ethylene inhibitors, oxygen radical scavengers and divalent cations. Cells can also be stabilized by subjecting the culture to a heat shock. Pretreated cells are acclimated to a reduced temperature and loaded with a cryoprotective agent such as DMSO, propylene glycol or polyethylene glycol. Loaded cells are incubated with a vitrification solution which, for example, comprises a solution with a high concentration of the cryoprotective agent. Vitrified cells retain less than about 20% water content and can be frozen at cryopreservation temperatures for long periods of time without significantly altering the genotypic or phenotypic character of the cells. Plant cells may also be cryopreserved by lyophilizing cells prior to exposure to a vitrification solution. The combination of lyophilization and vitrification removes about 80% to about 95% of the plant cell&#39;s water. Cells can be successfully cryopreserved for long periods of time and viably recovered. The invention also relates to methods for the recovery of viable plant cells from cryopreservation. Cells are thawed to about room temperature and incubated in medium containing a cryoprotective agent and a stabilizer. The cryoprotective agent is removed and the cells successfully incubated and recovered in liquid or semi-solid growth medium.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 08/486,204, filed Jun. 7, 1995.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for the cryopreservation ofplant cells and to methods for the recovery of plants cells which havebeen cryopreserved. The invention also relates to plants, viable plantcells and plant cells cultures which have been successfully recoveredfrom cryopreservation.

2. Description of the Background

Cryopreservation is based on the reduction and subsequent arrest ofmetabolic functions of biological material stored at ultra-lowtemperatures. Cryogenic preservation of plants and plant cells forextended periods without genetic change and the subsequent recovery ofnormal plant cells with unaltered characteristics and biosyntheticability has important implications in plant breeding, biomedicalresearch and genetic engineering. At the temperature of liquid nitrogen(-196° C.) almost all metabolic activities the cell ceases and cells canbe maintained in this suspended, but viable state for extended periods.

Plant cells are cryopreserved to avoid loss by contamination, tominimize genetic change in continuous cell lines, and to avoid aging andtransformation in finite cell lines. Traditional methods forpreservation of a desirable plant characteristic involve establishmentof colonies of plants in the field because many plants do not breed truefrom seeds. These field plant depositories demand large inputs of laborand land and incur high risks of loss due to weather, disease or otherhazards. An alternative to a field colony is the establishment of an invitro collection of plant tissue under normal or limited growthconditions. For long-term storage, elimination of routine subculturingis desirable because of concerns with mutation, contamination, laborcost and risk of human error associated with tissue culture.

Most biological materials, including plants, cannot survive freezing andthawing from cryogenic temperatures without cryoprotective agents andprocedures. A number of cryopreservatives possess properties which canprotect a cell from the damaging effects of cryogenic freezing. Theessence of cryopreservation is to effect cell dehydration andconcentration of the cytosol in a controlled and minimally injuriousmanner so that ice crystallization in the cytosol is precluded orminimized during, for example, quenching in liquid nitrogen.

In conventional cryopreservation procedures, cell dehydration iseffected by freeze-induced concentration of the suspending medium.Deleterious effects of dehydration are mitigated by the presence ofcryoprotective agents. Specimens such as cells and organs areequilibrated in a solution containing a cryopreservation agent such asdimethylsulfoxide (DMSO) or ethylene glycol. The suspension is cooledand seeded with an ice crystal at a temperature slightly below itsfreezing point. The suspension is cooled again at an optimum rate to anintermediate sub-zero temperature such as between about -30° C. andabout -40° C. and finally quenched in liquid nitrogen.

While routine cryogenic preservation of microorganisms, zygotes andanimals derived from zygotes is possible, the cryopreservation of plantcells is far from routine and often, different protocols for individualspecies of plants are necessary.

Taxus trees produces taxol, a diterpenoid alkaloid originally isolatedfrom the bark of the Pacific yew, Taxus brevifolia (M. C. Wani et al.,J. Am. Chem. Soc. 93:2325-27, 1971). Experiments have demonstrated thatthis compound effectively inhibits the polymerization of microtubules ofmammalian cells without undue toxicity and, as such, is an effectiveanti-tumorigenic agent. Clinical trails identified taxol as extremelyeffective against refractory ovarian, breast and other cancers. As such,taxol is a breakthrough in chemotherapy because of its rather unique,but basic mechanism of action which is fundamentally distinct from thatof conventional chemotherapeutic agents (L. A. Rowinsky et al., J. Natl.Cancer Instit. 82:1247-59, 1990).

The most daunting variable in the taxol equation so far is supply. Ittakes three to six, 100 year old Pacific yews to treat one patientbecause average yields of taxol are low (Witherup et al., 1990). Theproduction of an amount of taxol needed for treatment and testing willrequire the destruction of tens of thousands of yews. The yew populationhas been rendered nearly extinct by logging and as the number of Pacificyews dwindles, medical research must look for other forms of supply fortaxol. The usefulness of taxol, as well as many other compounds whichmay be propagated or harvested in plant cells, has fueled an interest inculturing taxus and other plant cells.

The culturing of plant cells for their biosynthetic ability posesspecial problems for current technology. Prolonged culturing of plantcells often results in a loss of biosynthetic ability which had beenpresent in the original isolates (Dhoot et al., Ann. Bot. 41:943-49,1977; Barz et al., Ber. Dtsch. Bot. Ges. 94:1-26, 1981). Phenotypicalterations also arise which further complicate cell culturing. Aprotocol for freezing plant cells, especially taxus cells, is animportant step in the development of biosynthetic methods for productionof useful plant alkaloids such as taxol.

SUMMARY OF THE INVENTION

The present invention overcomes the problems and disadvantagesassociated with current strategies and designs and provides novelmethods for cryopreservation and for the recovery of viablecryopreserved plant cells.

One embodiment of the invention is directed to methods for thecryopreservation of plant cells. Plant cells, which may be gymnospermsor angiosperms, are pretreated with a cryoprotective agent and astabilizer, and acclimated to a reduced temperature. Stabilizers suchas, for example, divalent cations, in part, protect cellular membranesfrom rupture. Pretreatment may also include an ethylene inhibitor, suchas an ethylene action inhibitor or an ethylene biosynthesis inhibitor.Pretreated plant cells are vitrified and frozen at a cryopreservationtemperature. Divalent cations may also be included in the vitrificationstep or a loading step. Acclimated cells are loaded with a loading agentwhich may be the same as the vitrifying agent and the loaded cellsvitrified with a vitrification solution. Vitrified plant cells arefrozen at cryopreservation temperatures, such as, between about -70° C.to about -200° C. or less.

Another embodiment of the invention is directed to methods forcryopreserving plant cells. Plant cells to be cryopreserved arepretreated with a cryoprotective agent, an ethylene inhibitor, divalentcations or heat-shock protein, and acclimated to a reduced temperature.The ethylene inhibitor may be an ethylene action inhibitor or anethylene biosynthesis inhibitor. Acclimated plant cells are vitrifiedand frozen at a cryopreservation temperature.

Another embodiment of the invention is directed to methods forcryopreserving plant cells. Plant cells to be cryopreserved are culturedin media comprising a vitrifying agent and a stabilizer at a reducedtemperature for a first period of time. The cultured plant cells arefurther cultured in media containing an increased concentration of thevitrifying agent for a second period of time. Plant cells vitrified inthe higher concentration of vitrifying agent are frozen at acryopreservation temperature.

Another embodiment of the invention is directed to methods forcryopreserving plant cells. Plant cells to be cryopreserved arelyophilized by vacuum evaporation and vitrified in a vitrifyingsolution. Lyophilization removes about 60% of the water from the cellsand in combination with vitrification can remove up to about 95%. Thevitrified and lyophilized plant cells are frozen and stored at acryopreservation temperature by, for example, quenching the cells intoliquid nitrogen.

Another embodiment of the invention is directed to methods forcryopreserving plant cells. Plant cells to be cryopreserved arepretreated with a heat shock. Heat shock induces the expression ofproteins that, in part, stabilize cellular membranes from rupture.Pretreatment may also include an ethylene inhibitor, such as an ethyleneaction inhibitor or an ethylene biosynthesis inhibitor. Pretreated plantcells are vitrified and frozen at a cryopreservation temperature.Divalent cations may also be included in the pretreatment orvitrification steps, or in a loading step.

Another embodiment of the invention is directed to methods forrecovering plant cells from cryopreservation. Plant cells arecryopreserved according to the methods of the invention. Thawed plantcells are warmed to a temperature above freezing and incubated in amedia comprising a cryoprotective agent and a stabilizer. The osmoticagent is removed and viable plant cells recovered.

Another embodiment of the invention is directed to methods forrecovering plant cells from cryopreservation. Plant cells arecryopreserved according to the methods of the invention. Thawed plantcells are warmed to a temperature above freezing and incubated in amedia comprising ethylene inhibitors, oxygen radical scavengers,divalent cations, cryoprotective agents or combinations of thesesubstances. Viable plant cells are recovered that show vigorous recoveryregrowth and can be quickly established into cell suspensions.

Another embodiment of the invention is directed to methods forrecovering cryopreserved plant cells from cryopreservation.Cryopreserved plant cells are thawed to a temperature above freezing andincubated in media comprising a cryoprotective agent and a stabilizer.The cryoprotective agent is removed such as by dilution of the mixtureor pelleting of the cells and viable plant cells recovered.

Another embodiment of the invention is directed to viable plant cellswhich have been cryopreserved by the method of the invention.Cryopreserved plant cells are not significantly genetically orphenotypically altered by cryopreservation.

Another embodiment of the invention is directed to methods forrecovering cryopreserved plant cells in suspension. Cryopreserved plantcells are thawed to a temperature above freezing. Thawed plant cells areincubated in liquid suspension and viable cells recovered in liquidmedia without a need for solid or semi-solid culture.

Another embodiment of the invention is directed to viable plants andplant cells cryopreserved and to viable plants and plant cells recoveredby the methods of the invention. Cells are not significantlygenotypically or phenotypically altered by the cryopreservation processand have a high proportion of survival.

Other embodiments and advantages of the invention are set forth, inpart, in the description which follows and, also in part, will beobvious from this description or may be learned from the practice of theinvention.

DESCRIPTION OF THE FIGURES

FIGS. 1 (A, B and C) Schematics of various cryopreservation and recoveryprotocols.

FIG. 2 Biosynthetic pathways of ethylene production and points ofinhibition.

FIG. 3 Procedure for cryopreservation of Taxus cells.

FIG. 4 Biomass increase in a Taxus chinensis suspension culture lineK-1.

FIG. 5 Chromatograms of (A) cells cryopreserved for 6 months incomparison with (B) non-cryopreserved cells.

FIG. 6 Chromatograms of (A) cells cryopreserved for 6 months incomparison with (B) non-cryopreserved cells.

FIG. 7 Southern blot analysis of the genetic stability of cryopreservedcells.

FIG. 8 PCR analysis of the genetic stability of cryopreserved cells.

DESCRIPTION OF THE INVENTION

As embodied and broadly described herein, the present invention isdirected to methods for the cryopreservation of plant cells, methods forthe recovery of cryopreserved plant cells and viable plant cells whichhave been successfully recovered from cryopreservation.

Plant cells are increasing useful for the production of recombinantprotein or unique products and chemical agents which are specific toplants or to the enzymatic pathways of plant cells. Plant cells such ascallus cultures can be maintained in a continuous state through repeatedsub-culturing. However, sub-culturing frequently results in increasedploidy, an increased risk of contamination, an accumulation ofspontaneous mutations, a decline and loss of morphogenetic potential, areduction of biosynthetic capacity for product formation, a reversion ofselected lines to wild-types, aging and transformation infinite celllines and unintentional selection of undesirable phenotypes. Each ofthese factors can severely impede the exploitation of cell culturesystems for commercial production of valuable compounds.

While animal tissue cultures cells have been routinely cryopreserved formany years, similar cryopreservation techniques for plant cells hasproven to be far more difficult. Plant cells and, in particular, plantcells in culture, exhibit an array of heterogeneity with respect togrowth rate, doubling time, mitotic index, cell synchrony, nuclear tocytoplasmic ratio and extent of vacuolation. Cells present in any giveculture also exhibit a variety of physiological and morphologicalvariations. Further, plant cell suspensions and adherent cell culturesrequire different protocols for cryopreservation. In addition, ifperformed improperly, cryopreservation can induce the very mutationswhich the process is attempting to prevent.

Surprisingly, it has been discovered that by using a series of steps andspecific agents, plant cells of most any genus and species can becryopreserved and successfully recovered. These methods are based on theobservations that successful plant cell cryopreservation involves theremoval of substantial amounts of water from the cells and that underappropriate conditions, significant amounts of water can be removedwithout seriously effecting cell viability. Cryopreservation protocolsdeveloped are highly successful at storing, maintaining and retrievingviable cells in a routine and reproducible manner. These protocols canbe established in routine unit operations to create germplasm storageand cell bank management systems. In addition, cells can be recoveredentirely in liquid suspension, a process previously thought not to bepossible with plant cultures. Products harvested from cryopreservedcells do not significantly vary from the original or parent cell asthere has been little if any phenotypic or genotypic drift, particularlywith respect to respect to growth and viability, product formation andcell biomass proliferation. Variances are determined from observable orquantifiable phenotypic or morphological alterations. Those alterationsbecome significant when they decrease the phenotypic parameter by morethan a small degree.

One embodiment of the invention is directed to methods for thecryopreservation of plant cells. These methods are surprisinglyreproducible and applicable to many types of plant cells. As such, theywill be markedly useful for the production of materials which requiregovernment or agency standards of reproducibility.

The basic process involves the removal of substantial amounts of waterfrom the cell by a combination of pretreatment (e.g. preculture withcryopreservant or stabilizers such as divalent cations, radicalscavengers or heat shock), cold acclimating, stepwise or single doseloading or vitrifying, lyophilizing, freezing and thawing steps. A widevariety of combinations of these steps is possible and every step is notnecessarily required for the successful cryopreservation and recovery ofviable plant cells that retain the characteristics and growth propertiesthat made them desirable. Some of the possible combinations of thevarious embodiments of the steps are schematically depicted in FIGS. 1A,1B and 1C, although it is understood that these variations are onlyexemplary. Each type of plant (e.g. class, order, family, genus,species, subspecies or variety) will more than likely have its own setof preferred conditions that will maximize recovery fromcryopreservation. From the selected variations disclosed herein, optimalparameters for cryopreservation can be easily determined.

Most any plant cell can be successfully cryopreserved and recoveredusing these processes including the gymnosperms and the angiosperms.Specific types of gymnosperms which can be cryopreserved include speciesof the genera Abies (firs), Cypressus (cypresses), Ginkgo (maidenhairtree), Juniperus (juniper), Picea (spruce), Pinus (pine), Pseudotsuga(Douglas fir), Sequoia, Taxus (yew), Tsuga (hemlock) or Zamia (cycad).Some of the more useful species of Taxus include T. baccata, T.brevifolia, T. canadensis, T. chinensis, T. cuspidata, T. floridana, T.globosa, T. media, T. nucifera and T. wallichiana. Angiosperms which canbe preserved include monocotyledon plant cells and dicotyledon plantcells. Monocotyledon plant cells include a variety of species of thegenus Avena (oat), Cocos (coconut), Dioscorea (yam), Hordeum (bareley),Musa (banana), Oryza (rice), Saccharum (sugar cane), Sorghum (sorghum),Triticum (wheat) and Zea (corn). Dicotyledon plants include species ofthe genus Achyrocline, Atropa, Brassica (mustard), Berberis, Sophora,Legume, Lupinus, Capsicum, Catharanthus, Conospermum, Datura, Daucus(carrot), Digitalis, Echinacea, Eschscholtzia, Glycine (soybean),Gossypium (cotton), Hyoscyamus, Lycopersicum (tomato), Malus (apple),Medicago (alfalfa), Nicotiana, Panax, Pisum (pea), Rauvolfia, Ruta,Solanum (potato) and Trichosanthes.

Plant cells may be freshly harvested specimens from the field as newgrowth needles, leaves, roots, bark, stems, rhizomes, callus cells,protoplasts, cell suspensions, organs or organ systems, meristems suchas apical meristems, seeds or embryos. Generally, low passage cells andprimary cultures show greater ultimate viability in culture or uponrecovery from cryopreservation. Alternatively, sample cells may beobtained from established in vitro cultures. Cultures may have been longestablished or only recently adapted to in vitro conditions of, forexample, specific temperatures, particular light intensities or specialgrowth or maintenance mediums. Such cells may be maintained assuspension cells or by growth on semi-solid nutrient medium.

Suspension cultures can be derived from callus cultures of a Taxusspecies or from thawed cryopreserved cells of a Taxus species. Lowpassage primary cell lines are very valuable to preserve as thesecultures may exhibit unique characteristics which are lost with extendedtime in culture. Many of these cell lines express diterpenoids such asthe diterpenoid alkaloid taxane, the ester side chain modified taxane,taxol (molecular weight 853), and a variety of other modifications oftaxane (baccatin or 10-deactylbaccatin).

Taxus cells for culture can be obtained from any plant material.Collections can be made from over North America as well as othercontinents. Tissue from any part of the plant including bark, cambium,needles, stems, seeds, cones and roots, can be selected and adapted forcell culture. Needles and meristematic regions of plants, especially oneto three month old new growth needles are preferred for initiating cellcultures. For example, selected plant tissue is surface-sterilized byimmersion in a four liter 10% solution of bleach with 10 drops of Tween20 added for 20 minutes. Cut tissues explants to very small sizes andcultured.

Taxus cultures typically exhibit variability in growth morphology,productivity, product profiles and other characteristics. As individualcell lines vary in their preferences for growth medium constituents,many different growth media may be used for induction and proliferationof the cell culture. Methods of sterilization, initiation of callusgrowth, and suspension growth, as well as suitable nutrient media, arewell-known to those of ordinary skill in the art.

Taxus suspension cultures are capable of rapid growth rates and highcell densities. Initial cultures of various Taxus species aresub-cultured by transfer into suitable media containing, for example,macro and micro nutrients, organic salts and growth hormones. Liquidcultures are exposed to air and preferably agitated to aerate the mediumor air may be bubbled in the medium. Cultures are maintained at atemperature of about 20° C. and at a pH of between about 3 to about 7,and preferably of between about 4 to about 6. Such cultures can beharvested by removal of the growth medium, for example, by filtration orcentrifugation. Cells with the highest viability are those at the earlylag or early cell division growth phases or recently passed through celldivision or mitosis. Generally, 4-10 day old cell suspension of a Taxusspecies in growth medium, preferably 5-8 day old cell suspensions ingrowth medium, and more preferably 6-7 day old cell suspensions of aTaxus species in growth medium, are suitable for use.

Each of the basic steps of cryopreservation are presented below:

Pretreatment

Plant cells to be cryopreserved can be pretreated with agents thatincrease cellular viability by removing harmful substances secreted bythe cells during growth or cell death from the culture medium. Theseagents, referred to as stabilizers herein, include substances that maybe naturally occurring or artificially produced and can be introduceddirectly into the culture medium. Stabilizers include anti-oxidants orradical scavenger chemicals that neutralize the very deleterious effectsattributable to the presence of active oxygen species and other freeradicals. Such substances are capable of damaging cellular membranes,both internal and external membranes, such that cryopreservation andrecovery are seriously compromised. If these substances are not removedor rendered otherwise ineffective, their effects on viability arecumulative over time severely limiting practical storage life.Furthermore, as cells die or become distressed, additional harmfulsubstances are released increasing the damage and death of neighboringcells.

Useful stabilizers include those chemicals and chemical compounds whichsequester highly reactive and damaging molecules such as oxygenradicals. Specific examples of these radical scavengers andanti-oxidants include reduced glutathione, 1,1,3,3-tetramethylurea,1,1,3,3-tetramethyl-2-thiourea, sodium thiosulfate, silver thiosulfate,betaine, N,N-dimethylformamide, N-(2-mercaptopropionyl) glycine,β-mercaptoethylamine, selenomethionine, thiourea, propylgallate,dimercaptopropanol, ascorbic acid, cysteine, sodium diethyldithiocarbomate, spermine, spermidine, ferulic acid, sesamol,resorcinol, propylgallate, MDL-71,897, cadaverine, putrescine, 1,3- and1,2-diaminopropane, deoxyglucose, uric acid, salicylic acid, 3- and4-amino-1,2,4-triazol, benzoic acid, hydroxylamine and combinations andderivatives of such agents.

Another group of stabilizers include agents that hinder or substantiallyprevent ethylene biosynthesis and/or ethylene action. Certain of thesecompounds also have scavenger properties as well. Effects of ethyleneinhibitors are substantial when the expression or action of ethylene issufficiently affected so as to increase cell recovery in thecryopreservation process. It is well known that many plant cells emitethylene when stressed. Ethylene damages cells and leads to cell death.Prevention of either the generation of ethylene or the action ofethylene will further enhance cell viability and cell recovery from thecryopreservation process.

As shown in FIG. 2, there are a large number of pathways in thebiosynthesis of ethylene and a equally large number of inhibitors. Forexample, biosynthetic pathways can be inhibited at the conversion ofS-adenosyhnethionine (SAM) (ACC synthase+SAM→ACC), aminocyclopropanecarboxylic acid (ACC) (ACC+ACC oxidase→ethylene), and ethylene(ethylene+ethylene oxidase→CO₂). A number of the biosynthetic inhibitorswhich can be used in the methods of the invention are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Inhibitors of Ethylene Biosynthesis                                           ______________________________________                                        Rhizobitoxin                                                                              Methoxylamine HCl                                                                            Hydroxylamine                                          Analogs                                                                     α-Canaline DNP (2,4-dinitrophenol) SDS (sodium lauryl                     sulfate)                                                                    Triton X-100 Tween 20 Spermine                                                Spermidine ACC Analogs α-Aminoisobutyric                                  Acid                                                                        n-Propyl Gallate Benzoic Acid Benzoic Acid                                      Derivatives                                                                 Ferulic Acid Salicylic Acid Salicylic Acid                                      Derivatives                                                                 Sesamol Cadavarine Hydroquinone                                               Alar AMO-1618 BHA (butylated                                                    hydroxyanisol)                                                              Phenylethylamine Brassinosteroids P-                                            chloromercuribenzoate                                                       N-ethylmaleimide Iodoacetate Cobalt Chloride and                                other salts                                                                 Bipyridyl Amino (oxyacetic) Acid Mercuric Chloride                              and other salts                                                             Salicyl alcohol Salicin Nickle Chloride and                                     other salts                                                                 Catechol Pffloroglucinol 1,2-Diaminopropane                                   Desferrioxamine Indomethacin 1,3-Diaminopropane                             ______________________________________                                    

There are also a large number of inhibitors of ethylene action. Some ofthese compounds are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        Inhibitors of Ethylene Action                                                 ______________________________________                                        Silver Salts     Benzylisothiocyanate                                           8-Hydroxyquinoline sulfate 8-Hydroxyquinoline citrate                         2,5-norbornadiene N-ethoxycarbonyl-2-ethoxy-1,2-                               dihydroquinoline                                                             Trans-cyclootene 7-Bromo-5-chloro-8-                                           hydroxyquinoline                                                             Cis-Propenylphosphonic Acid Diazocyclopentadiene                              Methylcyclopropane 2-Methylcyclopropane Carboxylic                             Acid                                                                         Methylcyclopropane carboxylate Cyclooctadiene                                 Cyclooctodine (Chloromethyl) Cyclopropane                                   ______________________________________                                    

It has been known since 1976 that silver ions act as a potentanti-ethylene agent in various plants and for improving the longevity ofplant tissues and cell cultures. For example, the longevity of cutcarnations can be increased by pretreatment with silver salts. Due tothe relative immobility of the silver ion, a basal treatment of the stemis considered to be less effective than a direct spray treatment of theflowers. The low mobility of silver ion is found to increase by cheatingthe metal to an anionic complex such as silver thiosulfate. Thiosulfatealone neither affects ethylene biosynthesis nor inhibits ethyleneaction.

Silver ion mediated ethylene inhibition could be explained on the basisthat silver is substituted for copper on the same receptor site. it isalso proposed that copper is the metal involved in enzymatic reactionsrelated to the biosynthesis or the action of ethylene. The similarity insize of silver and copper, the same oxidation state and the ability ofboth metals to form complexes with ethylene lend credence to this idea.

Silver salts including silver thiosulfate inhibits ethylene action inplants and plant cell cultures even though it has a remarkablestimulatory effect on its synthesis. The stimulatory effect of silverions (active ingredient of silver thiosulfate) on both ACC and ethylenebiosynthesis suggests increased conversion of ACC to ethylene due toincreased activity of ACC oxidase. Some of the silver salts that inhibitethylene action are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        Selected Silver Salts                                                         ______________________________________                                        Silver Thiosulfte                                                                          Silver Nitrate                                                                             Silver Chloride                                       Silver Acetate Silver Phosphate Citric Acid Tri-                                Silver Salt                                                                 Silver Benzoate Silver Sulfate Silver Oxide                                   Silver Nitrite Silver Cyanate Lactic Acid Silver                                Salt                                                                      Silver Pentafluoropropionate                                                                      Silver Hexafluorophosphate                                  Silver Salts of Toluenesulfonic Acid                                        ______________________________________                                    

Stabilizers are preferably incubated with plant cells prior to freezing,although their presence during the freezing process, recovery, thawingand regrowth, may also be desirable. For example, ethylene biosynthesisinhibitors and ethylene action inhibitors may be more desirable to havein the culture medium during thawing and regrowth. Incubations can beperformed for hours or days as the agents themselves are generally notharmful to the cells and may even increase viability. Some of the moresensitive plant cell lines may require longer treatments while othersshorter. The exact period of incubation can be easily determinedempirically. Preferably, plant cells are cultured in growth medium withthe stabilizer or a combination of stabilizers for about 1 to about 10days, more preferably for about 1 to about 7 days and even morepreferably about 3 days. This amount of time is typically sufficient fordamaging substances in the medium to be eliminated or at least reducedto levels which are no longer harmful to the cells.

Incubations can be performed in liquid or semi-solid mediums such asgrowth medium, medium that encourages metabolism and cell proliferation,or maintenance medium, medium that provides a balance of salts andnutrients without necessarily encouraging cell growth. As the cells arebeing prepared for cryopreservation, it is sometimes desirable toincubate in maintenance medium (a sub-optimal or slow growth culturemedium; e.g. one fourth of the salt concentration of the normal growthmedium) to reduce the metabolic processes of the cells.

Pretreatment can be performed by preculturing cells at room temperatureor at temperatures which the plant cells are typically cultured.Preferably, preculturing is performed at about room temperature (20° C.)for ease of handling and as most plant cells are fairly stable at roomtemperature. Stabilizers can be added directly to the medium andreplenished as necessary during the pretreatment process. Stabilizerconcentrations are particular to specific stabilizers, but are generallyused at between about 1 μM to about 1 mM, or preferably at between about10 μM to about 100 μM, although more or less of one or more stabilizingagents would not be uncommon.

Pretreatments may also involve incubating cells in the presence of oneor more osmotic agents. Examples of useful osmotic agents include sugarssuch as saccharides and saccharide derivatives, amino or imino acidssuch as proline and proline derivatives, or combinations of theseagents. Some of the more useful sugars and sugar derivatives arefructose, glucose, maltose, mannitol, sorbitol, sucrose and trehalose.Osmotic agents are utilized at a concentration that prepares cells forsubsequent loading, lyophilization and/or vitrification. Concentrationscan vary greatly between different agents, but are generally betweenabout 50 mM to about 2 M. These concentrations can be achieved by addingsmall amounts of the agents continuously or incrementally over time(stepwise) until a desired level is achieved. Preferably, osmotic agentsconcentration in media are between about 0.1 M to about 0.8 M, and morepreferably at between about 0.2 M to about 0.6 M. Alternatively, theosmotic agent is employed as an aqueous solution at a concentration ofbetween about 1% to about 10%, by weight.

Pretreatment may also simply involve the addition of membranestabilizers such as compounds that intercalate into the lipid bilayer(e.g. sterols, phospholipids, glycolipids, glycoproteins) or divalentcations into the cell culture, the loading solution and/or thevitrification solution. Divalent cations stabilize heat shock andmembrane proteins, and also reduce electrostatic repulsion betweencellular and other membranes. Magnesium, manganese and calcium arepreferred choices as inexpensive and readily available divalent cationsin the form of, for example, CaCl₂, MnCl₂ and MgCl₂. Sodium is lesspreferred due to its toxicity at any more than trace concentrations.Preferred concentrations range from about 1 mM to about 30 mM, and morepreferably from about 5 mM to about 20 mM and still more preferably atabout 10 mM or 15 mM. In addition, the presence of divalent cations inthe medium reduces freezing temperatures and allows for the more rapidpassage of cells through freezing points. The temperature of bothintercellular and intracellular ice crystal formation is reduced uponfreezing and also during thawing. The presence of these agents duringthawing and post-thawing culture can also be important.

Cold Acclimation

During or at some time after pretreatment, cells to be cryopreserved maybe acclimated to a temperature which is reduced from culturingtemperatures, but above freezing. This prepares cells for thecryopreservation process by significantly retarding cellular metabolismand reducing the shock of rapid temperature transitions through some ofthe more critical temperature changes. Critical temperature ranges arethose ranges at which there is the highest risk of cell damage, forexample, around the critical temperatures of ice crystal formation. Asknown to those of ordinary skill in the art, these temperatures varysomewhat depending upon the composition of the solution. For water, theprincipal component of most cell culture mediums, ice crystal formationand reformation occur at about 0° C. to about -50° C.

Acclimation results in the accumulation of endogenous solutes thatdecreases the extent of cell dehydration at any given osmatic potential,and contributes to the stabilization of proteins and membranes duringextreme dehydration. In addition, cold adaptation interactssynergistically with the vitrifying agents and results in alterations inthe liquid conformation of the cellular membranes, that increasetolerance to both osmotic exclusions and dehydration.

Acclimation may be carried out in a stepwise fashion or gradually. Stepsmay be in decreasing increments of about 0.5° C. to about 10° C. for aperiod of time sufficient to allow the cells acclimate to the lowertemperature without causing damage. The temperature gradient, whethergradual or stepwise, is scaled to have cells pass through freezingpoints as quickly as possible. Cells may be gradually, in a step-wise orcontinuous manner, or rapidly acclimated to the reduced temperature.Techniques for acclimation are well known to those of ordinary skill andinclude commercially available acclimators. Gradual acclimationcomprises reducing incubation temperatures about 1° C. per hour untilthe target temperature is achieved. Gradual acclimation is most usefulfor those cells considered to be most sensitive and difficult tocryopreserve. Stepwise acclimation comprises placing the cells in areduced temperature for a period of time, a further reduced temperaturefor another period of time. These steps are repeated as required.

Suspension cells can be in late lag or early cell division phases toachieve the greatest survival rates on freezing and thawing. Cellsbeyond these phases exhibit higher degrees of vacuolation anddifferentiation and are larger in size, thus enhancing the risk offreezing injury and decreasing survival rates on freezing and thawing.

Loading

Loading involves the equilibration of cells in a solution of one or morecryoprotectants. Agents utilized during loading may be referred to asloading agents. Useful loading agents may include one or moredehydrating agents, permeating and non-permeating agents, and osmoticagents. Suitable agents for loading include agents which induce celldehydration upon introduction to a cell suspension. Both permeatingagents such as DMSO and ethylene glycol, and a combination of permeatingand nonpermeating osmotic agents such as fructose, sucrose or glucose,and sorbitol, mannitol or glycerol can be used. This step increasessolute concentration of the cytosol by introducing moderateconcentrations of cryoprotective agents, generally at between about 0.5M to about 2 M or between about 5% to about 20%, by weight, into thecytosol. To minimize the time required for equilibration, loading isusually performed at about room temperature, although optimaltemperature and other conditions for loading will preferably matchconditions such as medium, light intensities and oxygen levels thatmaintain a viable cell culture.

In the loading step, single cryoprotective agents or combinations ofdifferent cryoprotective agents can be added directly to the incubationmedium continuously or in a stepwise fashion. Stepwise loading issometimes desired to facilitate delivery of the loading agent to cellsas it is somewhat more gentle than single dose loading. Time incrementsor interval between additions for stepwise loading may range fromminutes to hours or more, but are preferable from about one to about tenminutes, more preferably from about one to about five minutes and stillmore preferably about one or about two minute intervals. The numbers ofadditions in a stepwise loading procedure is typically whatever ispractical and can range from very few to a large plurality. Preferably,there are less than about twenty additions, more preferably less thanabout ten and even more preferably about five. Interval periods andnumbers of intervals are easily determined by one of ordinary skill inthe art for a particular type of cell and loading agent. Cells areincubated in a solution containing the loading agent or agents (withcontinuously or stepwise increasing amounts of loading agent) toequilibrate intracellular and/or extracellular concentrations of theagent. Incubation times range from minutes to hours as practical. Theprotective effects of loading include, in part, removal of a small, butsignificant amount of water from the cell. This prepares the cell forsubsequent vitrification and/or lyophilization by minimizing the shockof sudden intracellular water loss.

After loading, growth medium containing the cryoprotective agent can beremoved or, if the following agent (vitrifying agent) to be utilized isthe same or a similar agent, the loading agent can remain and theconcentration simply increased for vitrification.

Vitrification

Cells to be cryopreserved are vitrified following pretreatment, loadingand/or lyophilization. There are several advantages of the verificationprocedures. By precluding ice crystal formation in the system, the needto optimize the complex set of variables which lead to ice formation iseliminated. Further, specimens can be plunged directly in liquidnitrogen, the procedure does not require extensive equipment requiredfor controlled cooling. The vitrification procedure also offers thegreatest potential for developing cryopreservation procedures forcomplex tissues and organs that are comprised of several different typesof cells.

Vitrification procedures involve gradual or stepwise osmotic dehydrationof the cells or specimens prior to quenching in liquid nitrogen. Invitrification procedures, cell dehydration is effected by directexposure to concentrated solutions prior to cooling in liquid nitrogen.Exposure can be gradual with continuously increasing amounts of thevitrification added to the cells or stepwise wherein increasing amountsare added over a set period of time. Time increments or interval betweenadditions for stepwise vitrification may range from minutes to hours ormore, but are preferable from about one to about ten minutes, morepreferably from about one to about five minutes and more preferablyabout one or about two minutes. The numbers of additions in a stepwisevitrification procedure is typically whatever is practical and can rangefrom very few to a large plurality. Preferably, there are less thanabout twenty additions, more preferably less than about ten and evenmore preferably about five. Interval periods and numbers of intervalsare easily determined by one of ordinary skill in the art for aparticular type of cell and vitrification agent. Cells are incubated ina solution containing the vitrification agent or agents (withcontinuously or stepwise increasing amounts) to equilibrateintracellular and/or extracellular concentrations of the agent asdesired. Incubation times vary from minutes to hours as practical. Underideal conditions the cells or organs can be cooled at extremely rapidrates without undergoing intercellular or intracellular ice formation.As a result, all of the factors that affect ice formation are obviatedand there are several practical advantages of the vitrificationprocedures in comparison to conventional cryopreservation procedures.Vitrification offers the greater potential for developingcryopreservation procedures for complex tissues and organs. Byprecluding significant ice formation in the system, the vitrificationprocedure is operationally more complex than conventionalcryopreservation procedures. Further, vitrification allows for the useof ultra-rapid cooling rates to circumvent problems of chillingsensitivity of some specimens. No specialized or expensive equipment isrequired for controlled cooling rates.

Vitrification is a cryogenic method wherein a highly-concentratedcytosol is super-cooled to form a solid, metastable glass withoutsubstantial crystallization. The major difficulty in cryopreservation ofany cell is the formation of intracellular ice crystals during bothfreezing and thawing. Excessive ice crystal formation will lead to celldeath due to disruption of cellular membranes and organelles. One methodto prevent ice crystal formation is to freeze the cells rapidly suchthat the ice crystals formed are not large enough to cause significantdamage. When a cell with a low water content is frozen rapidly, itvitrifies. Vitrification by rapid freezing is not possible with cellssuch as plant cells which containing a high water content. To vitrifyhigh water content cells, freezing point reduction agents and icecrystal inhibitors is needed in addition to rapid freezing forvitrification. A properly vitrified cell form a transparent frozenamorphous solid consisting of ice crystals too small to diffract light.If a vitrified cell is allowed to warm to about -40° C., it may undergodevitrification. In devitrification, ice crystals enlarge andconsolidate in a process which is generally detrimental to cellsurvival. Vitrification solutions enhance vitrification of cells uponfreezing or retard devitrification upon thawing.

Most cryopreservation solutions can transform the subject material intoa glass or glass-like material provided cooling rates are sufficientlyrapid to prevent the nucleation and growth of ice crystals, with thecritical cooling rate dependent on the solute concentration. Similarly,vitrification of the cells can be effected if the cytosol issufficiently concentrated. In cryopreservation procedures, this isachieved by dehydrating the cells in extremely concentrated solutionsprior to quenching in liquid nitrogen. In the cryopreservation process,the cytosol is concentrated to the level required for vitrification byplacing the specimens in a concentrated solution of a cryoprotectivesuch as the vitrifying agent. Concentrations such as about 4 M to about10 M, or between about 25% to about 60%, by weight, are preferred. Thisproduces an extreme dehydration of the sample cells. Solutions in excessof 7 M typically remove more than 90% of the osmotically active waterfrom the cells; however, precise concentrations for each agent can beempirically determined. Vitrifying agents which may be used includeDMSO, propylene glycol, glycerol, polyethylene glycol, ethylene glycol,butanediol, formamide, propanediol and mixtures of these substances.

Suitable vitrification solutions include culture medium with DMSO(1-50%), propylene glycol (about 5-25%), glycerol (about 0-50%),PEG-8000 (about 5-20%), ethylene glycol (about 10-75%), ethyleneglycol/sorbitol (about 60/20 weight percent to about 10/60 weightpercent), and ethylene glycol/sorbitol (about 40/30 weight percent).Ethylene glycol/sorbitol is preferred and can be employed atconcentrations of, for example, 50/30%, 45/35%, 40/40%, 40/30%, 30/50and, preferably, 30/40%. Such vitrification solutions can be utilized attemperatures from about 1° C. to about 8° C., preferably at atemperature of from 2° C. to 6° C., and more preferably at about 4° C.

To minimize the injurious consequences of exposure to vitrificationsolutions, dehydration may be performed at about 0° C. to about 4° C.,with the time of exposure as brief as possible. Under these conditions,there is no appreciable influx of additional cryopreservation into thespecimens because of the difference in the permeability coefficient forwater and solutes. As a result, the specimens remain contracted and theincrease in cytolic concentration required for vitrification is attainedby dehydration. However, equilibrium (loading) of the cells withcryoprotectants is not always required for successful vitrification ofplant cells or organs. For example, in some cases, preculturing withloading agents achieve the same purposes as the loading step.

In those instances in which loading is required, it primarily serves toprevent dehydration-induced destabilization of cellular membranes andpossibly proteins. However, in the absence of a loading step, there canbe less survival of cells following the dehydration and cooling/warmingsteps. Thus, intracellular ethylene glycol or other cryoprotectantsduring the loading step not only favors vitrification of the cellsduring cooling, but also protects cells against injury during thedehydration step.

Lyophilization

Lyophilization is directed to reducing the water content of the cellsbefore cryopreservation by vacuum evaporation. Vacuum evaporationinvolves placing the cells in an environment with reduced air pressure.Depending on the rate of water removal desired, the reduced ambientpressure operating at temperatures of between about -30° C. to -50° C.may be at 100 torr, 1 torr, 0.01 torr or less. Under conditions ofreduced pressure, the rate of water evaporation is increased such thatup to 65% of the water in a cell can be removed overnight. With optimalconditions, water removal can be accomplished in a few hours or less.Heat loss during evaporation maintains the cells in a chilled state. Bycareful adjustment of the vacuum level, the cells may be maintained at acold acclimation temperature during the vacuum evaporation process. Astrong vacuum, while allowing rapid water removal exposes the cells tothe danger of freezing. Freezing may be controlled by applying heat tothe cells directly or by adjustment of the vacuum level. When the cellsare initially placed in the evaporative chamber, a high vacuum may beapplied because the residue heat in the cells will prevent freezing. Asdehydration proceeds and the cell temperature drops, the vacuum may bedecreased or heating may be applied to prevent freezing. The semi-drycells may have a tendency to scatter in an evaporative chamber. Thistendency is especially high at the end of the treatment when anairstream is allowed back into the chamber. If the air stream proximatesthe semi-dry cells, it may cause the cells to become airborne and causecross contamination of the samples. To prevent such disruptions,evaporative cooling may be performed in a vacuum centrifuge wherein thecells are confined to a tube by centrifugal force while drying. Theamount of water removed in the process may be monitored periodically bytaking dry weight measurement of the cells. When the desired amount ofwater is removed, vitrification solution may be added directly to thesemi-dry cells for a period to time prior to direct freezing in liquidnitrogen.

Freezing

Plant cells, which may have been pretreated, loaded, vitrified and/orlyophilized, are preserved by freezing to cryopreservation temperatures.The freezing step should be sufficiently rapid to prevent the formationof large ice crystals which are detrimental to the cell's survival.Cells may be directly frozen, that is brought directly into contact withan agent already at cryopreservation temperature. Direct methods includedripping, spraying, injecting or pouring cells directly into a cryogenictemperature fluid such as liquid nitrogen or liquid helium. Cells mayalso be directly contacted to a chilled solid, such as a liquid nitrogenfrozen steel block. The cryogenic fluid may also be poured directly ontoa container of cells. The direct method also encompasses contactingcells with gases, including air, at a cryogenic temperature. A cryogenicgas stream of nitrogen or helium, may be blown directly over or bubbledinto a cell suspension. Indirect methods involve placing the cells in acontainer and contacting the container with a solid, liquid, or gas atcryogenic temperature. Proper containers include, for example, plasticvials, glass vials or ampules which are designed to withstand cryogenictemperatures. Containers for indirect freezing methods do not have to beimpermeable to air or liquid. For example, a plastic bag or aluminumfoil are adequate. Furthermore, the container may not necessarily beable to withstand cryogenic temperatures. A plastic vial which cracks,but remain substantially intact under cryogenic temperatures may also beused. Cells may also be frozen by placing a sample of cells on one sideof a metal foil while contacting the other side of the foil with a gas,solid, or liquid at cryogenic temperature. Freezing should besufficiently rapid to inhibit ice crystal formation. The freezing timeshould be around 5 minutes or 4 minutes, 3 minutes, 2 minutes, or oneminute or less. The critical freezing time should be measure from theframe of reference of a single cell. For example, it may take 10 minutesto pour a large sample of cells into liquid nitrogen, however theindividual cell is frozen rapidly by this method.

Thawing

Another embodiment of the invention is directed to methods for thawingcryopreserved cells. Proper thawing and recovery is essential to cellsurvival and to recovery of cells in a condition substantially the sameas the condition in which they were originally frozen. As thetemperature of the cryopreserved cells is increased during thawing,small ice crystals consolidate and increase in size in a process termeddevitrification. Large intracellular ice crystals are generallydetrimental to cell survival. To prevent devitrification, cryopreservedcells should be thawed as rapidly as possible. The rate of heating maybe at least about 30° C. per minute to 60° C. per minute. More rapidheating rates of 90° C. per minute, 140° C. per minute to 200° C. ormore per minute can also be used. While rapid heating is desired, plantcells have reduced ability to survive incubation temperaturesignificantly above room temperature. To prevent overheating, the celltemperature should be carefully monitored. Any heating method can beemployed including conduction, convection, radiation, electromagneticradiations or combinations thereof. Conduction methods involve immersionin water baths, placement in heat blocks or direct placement in openflame. Convection methods involve the use of a heat gun or an oven.Radiation methods involve, for example, heat lamps or ovens such asconvection or radiation ovens. Electromagnetic radiation involves theuse of microwave ovens and similar devices. Some devices may heat by acombination of methods. For example, an oven heats by convection and byradiation. Heating should be terminated as soon as the cells and thesurrounding solutions are in liquid form, which should be above 0° C.Cryopreserved cells are often frozen in the presence of one or moreagents that depress the freezing point. When these agents are present,the frozen cells may liquify at a temperature below 0° C. such as atabout -10° C., -20° C., -30° C. or -40° C. Thawing of the cryopreservedcells may be terminated at any of these temperature or at a temperatureabove 0° C.

Post-Thawing

Dilution of the vitrification solution and removal of cryopreservativefrom the cells, referred to as unloading, should be performed as rapidlyas possible and as quickly as possible subsequent to thawing of thecryopreserved cell sample. Due to the high intracellular concentrationsof cryopreservative, it is preferred to effect the dilution of thesuspending medium while minimizing osmotic expansion. Therefore,dilution of the suspending medium and efflux of the cryopreservativefrom within the sample specimen is usually accomplished by dilution inan hypertonic medium or a step-wise dilution.

Thawed cells can be gradually acclimated to growth conditions tomaximize survival. Vitrification agents may be cytotoxic, cytostatic ormutagenic, and should be removed from the thawed cells at a rate whichwould not harm the cells. A number of removal methods may be used suchas resuspension and centrifugation, dialysis, serial washing,bioremediation and neutralization with chemicals, or electromagneticradiation. The rapid removal of some vitrification solutions and osmoticagents may increase cell stress and death and thus the removal step mayhave to be gradual. Removal rates may be controlled by serial washingwith solutions that contain less osmotic or vitrification agents. Othermethod to reduce the removal rate include dialysis with less permeablemembranes, serial growth on semisolid or liquid media containing lessand less concentration of vitrification agents. Other methods includegradual dilutions, dialysis, bioremediation, neutralization andcatalytic breakdown of the cryogenic agent.

Thawing and post-thaw treatments may be performed in the presence ofstabilizers to ensure survival and minimize genetic and cellular damage.The stabilizer such as, for example, divalent cations or ethyleneinhibitors, reduce, eliminate or neutralize damaging agents whichresults from cryopreservation. Such damaging agents include freeradicals, oxidizers and ethylene. Some of these agents have multipleproperties and are very useful in this regard. Survival and regrowthrates are surprisingly enhanced with the addition of stabilizers duringthe thawing and post thawing steps.

Cells can be regrown in suitable media after levels of osmotic orvitrification agents are reduced to an acceptable level. One method forregrowth involves placing the thawed cells in semisolid growth media,such as agar plates, until a callus is formed. Cells may be recoveredfrom the callus and grown in liquid culture. Alternatively, callus cellsmay be induced to grow shoots and roots by placement in semisolid mediacontaining the appropriate hormones. Callus cells with shoots and rootsmay be gradually acclimated to grow on sterile soil in a greenhouseuntil a plant develops. The greenhouse plant may be acclimated to grownoutside a greenhouse in its natural environment. Alternatively, a cellmay be thawed and regrown without the use of semi-solid media. That is,after removal of osmotic and vitrification agents, the cells may beplaced directly into liquid media for regrowth.

Inclusion of one or more of the variations disclosed herein cansubstantially increase cell recovery after cryopreservation for avariety of cell types. These methods prevent problems associated withtoxicity due to cryoprotectants, build-up of ethylene, ethylene actionon specific targets including the membrane associated receptors,instability of membranes and membrane leakage. From the methodsdisclosed herein, particular combinations of steps have been identifiedas optimal for certain cell types. For example, tomato, potato andtobacco cells respond well to cryopreservation using stepwise loadingand/or vitrification with divalent cations (Protocols 10, 12, 13, 14).

One method to implement cryopreservation of plant cells, for example,taxol producing Taxus cells, is schematically represented in FIG. 3.Briefly, a callus cell growth from a primary isolate or from a cellculture is adapted for culture in liquid growth medium. After the cellshave adjusted to liquid culture, they are transferred to a pretreatmentgrowth medium containing an osmotic agent and a stabilizer for 24 to 72hours. Precultured cells are subsequently cold acclimated by theincubation of the pretreatment culture at 4° C. for 1 to 4 hours. Aftercold acclimation, cells are transferred to a centrifuge vial andsubjected to mild centrifugation which pellets the cells without damage.Supernatant, comprising the pretreatment media with osmotic agents andthe stabilizer, is aspirated from the cell pellet and discarded. Aprechilled solution, comprising a stabilizer and a vitrification agent,is added to the cell pellet and the cells are gently resuspended. After3 minutes of treatment with the vitrification solution, the cells areplaced into archival cryogenic storage by immersion of the vialcontaining the cells in liquid nitrogen. Cryogenically preserved cellsare stored in liquid nitrogen for a period from about 30 minutes to manyyears. To revive the cells, the vial containing the cryopreserved cellsis rapidly transferred from liquid nitrogen to a 40° C. water bath. Thevial is removed from the 40° C. bath when the contents are liquified. Analiquot of the thawed cells is inspected for immediate viability by dyeexclusion analysis. The remaining cells are washed by a series of coldgrowth medium containing a stabilizer and a progressively lowerconcentrations of osmotic agents. After sufficient osmotic andvitrification agents are removed from the cells by serial washes, thecells are transferred directly into liquid culture. Alternatively, cellsare placed on a plating paper and transferred to a series of semi-solidmedium with a stabilizer and a decreasing amount of osmotic andvitrification agents. This serial plating is continued until the cellshave adjusted to growth on semi-solid medium in the absence of osmoticand vitrification solutions.

Other methods of cell cryopreservation are disclosed in FIGS. 1A, 1B and1C. As can be see in this schematic representation, a number ofvariations are preferred. For example, in step 1, cell biomass isprecultured in liquid medium containing osmotic agent (sucrose, sorbitolor mannitol with concentration ranges from 0.06 M to 0.80 M) for 1 to 6days in liquid medium. In step 2, loading is performed stepwise.Precultured cell biomass is loaded with cryoprotectants (20% ofvitrification solution (V2N) containing ethylene glycol/sorbitol at aconcentration of 40/30 weight percent in nutrient culture medium or 20%of vitrification solution (VS3) containing 30% (w/v) glycerol, 15% (w/v)ethylene glycol and 15% (w/v) dimethylsulfoxide in nutrient culturemedium or in 0.4M sucrose solution (pH 5.6 to 5.8). Cell biomass inliquid is mixed gently and incubated on ice or in refrigerate at 0° C.to 4° C. for five minutes. Immediately after this step, theconcentration of cryoprotectants in liquid is increased stepwise byadding five times chilled 100% vitrification (V2N or VS3) solution atone minute intervals until the final concentration of vitrificationsolution is reached at 65%. The total time used for this loading stepand incubation at 0° to 4° C. is ten minutes. The mixture (cellbiomass+vitrification) is centrifuged for up to five minutes (one tofive minutes) at 100× g and the supernant is discarded.

Alternatively, in step 2, loading can be performed in a single step.Precultured cell biomass is loaded with cryoprotectants (65% of avitrification solution--V2N containing ethylene glycol/sorbitol at aconcentration of 40/30 weight percent in nutrient culture medium; or 65%of a vitrification solution--VN3 containing 30% (w/v) glycerol, 15%(w/v) ethylene glycol and 15% (w/v) DMSO in nutrient culture medium; orin 0.4 M sucrose solution (pH 5.6-5.8)). Cell biomass in liquidcontaining cryoprotectants is mixed gently and incubated on ice orrefrigerated at 0° C. to 4° C. for ten minutes.

Alternatively, loading can be performed stepwise or in one step withcryoprotectants containing one or more membrane stabilizers. Allbiological membranes have a common overall structure. They areassemblies of lipid and protein molecules held together by noncovalentinteractions. Lipids are arranged as a bilayer, which provides the basicstructure of the membrane and serves as a relatively impermeable barrierto the flow of most water-soluble molecules. Protein molecules arewithin the lipid bilayer and modulate the various functions of themembrane. Some service to transport specific molecules into or out ofthe cell such as, for example, channel proteins such as passive channelproteins, carrier proteins and proteins involved in active transport.Others are enzymes that catalyze membrane-associated reactions, andstill others serve as structural links between the cell's cytoskeletonand extracellular matrix, and/or as receptors for receiving transducingchemical signals from the cell's environment.

Certain plant species and cell lines are recalcitrant to previouslyestablished cryopreservation protocols, partly due to their sensitivityto the type and concentration of cryoprotectants. There are at least twopotential causes of membrane destabilization, chemical toxicity of thecryoprotectants used in the loading and dehydration steps and extremedehydration resulting from exposure to the concentrated solutions (e.g.vitrification). Other factors such as the ice formation during eithercooling or warming, and mechanical disruption of cells due to fracturingof the glass can also contribute to destabilization of membranes.

Severe dehydration resulting from the exposure to concentrated solutionsof cryoprotectants can cause the structural transitions in a diversearray of biological molecules including lipids, proteins and nucleicacids. Under these conditions, there are several alterations in theultrastructure of the plasma membrane and other cellular membranes.These changes include the formation of a particulate domains in theplasma membrane which result in the removal of water that is associatedwith the hydrophilic regions of the proteins and lipid components of themembranes. Alternatively, destabilization can also occur by contactingplant cell membranes with a permeablizing cryoprotectant compound (i.e.a compound that fluidizes a channel protein within a plant cellmembrane). Stepwise loading and/or vitrification can minimize membranedestabilization resulting from severe dehydration. Other featuresrelated to the substantial changes in cell volume, permeability of theplasma membrane, etc. can also be minimized by stepwise loading.

Heat-shock treatment will also stabilize membranes and proteins.Heat-shock treatment, after preculturing cell biomass with osmoticagents, is not necessary prior to loading cells with cryroprotectants.However, heat-shock treatment is required when the cell biomass is notprecultured with osmotic agents. Furthermore, it is important to loadcells with vitrifying solution (cryoprotectants) containing divalentcation (CaCl₂, MnCl₂ or MgCl₂ ; at 5 mM to 20 mM) (e.g. Protocols 11 and12). Heat-shock treatment is usually followed after preculturing of cellbiomass. It tends to improve the survival of cells aftercryopreservation by about 20% to about 40%. This procedure involves theincubation of cells or cell biomass (either precultured ornon-precultured) in a water-bath shaker at about 37° C. for up to aboutfour hours. After this treatment, cells in liquid medium (with orwithout osmotic agent) are transferred to room temperature (23° C. to25° C.) for up to four hours before being used for cryopreservation(loading step).

Heat-shock treatment is known to induce de novo synthesis of certainproteins (heat-shock proteins) that are supposed to be involved inadaptation to stress. Heat-shocked cells that are not precultured withosmotic agent do not survive freezing. Heat shock induces tolerance toexposure by abruptly increasing the concentration of osmotics in cellsthat result from freezing by the formation of heat-shock proteins thatstabilize proteins and membranes. Heat shock is performed by culturingthe cells in a water bath at between about 31° C. to about 45° C.,preferably between about 33° C. to about 40° C. and more preferably atabout 37° C. Culturing is performed from a few minutes to a few hours,preferably from about one hour to about six hours, and more preferablyfrom about two hours to about four hours.

Divalent cations such as CaCl₂, MnCl₂ or MgCl₂, can also be included ina vitrifying solution prior to the exposure of cells to LN₂temperatures. Divalent cations included in the vitrifying solutionsappear to stabilize plant cell membranes and heat-shock proteins.Divalent cations stabilize cell membranes by reducing electrostaticrepulsion between ionic centers on the membrane. Additionally, divalentcations can also prevent ice nucleation during freezing and thawing.Other compound such as sterols, phospholipids, glycolipids orglycoproteins which intercalate into the lipid bilayer of membranes, mayalso effectively stabilize plant cell membranes. These compounds may besubstituted for divalent cations in stabilization of plant cellmembranes and/or heat-shock proteins.

The procedure for stepwise loading precultured or non-precultured cellbiomass (with or without heat-shock treatment at 37° C. up to fourhours) with cryoprotectants is the same as before except the vitrifyingsolutions (V2N or VS3) contained the membrane stabilizer divalentcations (e.g. CaCl₂, MnCl₂, MgCl₂) at a concentration ranging from 5 mMto 20 mM.

In step 3, the vitrification step can be stepwise. Cells or cell biomassthus obtained after loading and centrifugation are further treated withvitrifying solution (V2N or VS3) in a stepwise manner. This step usuallyinvolves the transfer of cell biomass after centrifugation to a chilledcryovial, and adding a concentrated (100%) vitrification solution at oneminute intervals during the three minute incubation period at 0° C. Atthe end of incubation period, the contents (cells+vitrifying solution)in a cryovial are mixed gently prior to a plunge into LN₂.Alternatively, vitrification can be performed in a single step. Cells orcell biomass thus obtained after loading and centrifugation are furthertreated with vitrification solution (100%). This step usually involvesthe transfer of cell biomass after centrifugation to a chilled cryovial,and adding a concentrated (100%) vitrifying solution. The contents in acryovial are mixed gently and are incubated for three minutes at 0° C.prior to a plunge into LN₂. Alternatively still, vitrification can bestepwise or one step with cryoprotectants containing divalent cationsuch as CaCl₂ or MgCl₂.

The procedure for vitrification of loaded cell biomass withcryoprotectants is the same as before except the concentrated vitrifyingsolution (V2N or VS3) contained the membrane stabilizer divalent cations(CaCl₂ or MgCl₂) at a concentration ranging from about 5 mM to about 20mM.

Freezing of cells is generally rapid in cryovials in LN₂ or the rapidtransfer of cryovials containing cells in vitrification solution to astorage vessel containing liquid nitrogen (LN₂).

Thawing involves a rapid warming (e.g. about 20 seconds to about 45seconds) of cryovials containing frozen cell biomass or cells in a waterbath set at 40° C. or 60° C. Post-thawing involves the immediatetransfer of liquefied cell biomass in cryovials to a sterile centrifugetube containing liquid nutrient medium with an osmotic agent (sucrose,sorbitol or mannitol; concentration range 1M to 2M) and an ethyleneinhibitor or ethylene action inhibitor (silver thiosulfate;concentration range from about 5 μM to about 20 μM). Contents are mixedgently and incubated on a shaker (120 rpm) for 30 minutes at roomtemperature. This is followed by centrifugation and transferring thecells in a pellet to two layers of sterile filter paper placed on ablotting paper (to aid in removal of excessive moisture from cells orcell biomass). Cells or cell biomass was incubated for two minutes.After this incubation period, the upper filter paper with cells istransferred sequentially to solid nutrient medium containing osmoticagent at a decreased concentration from 0.8M to 0.1M sucrose (to bringabout the osmotic adjustment of cells). At each step, cells on a filterpaper are incubated for 1/2 hour to 1 hour and finally transferred to anormal solid nutrient medium without the presence of any additionalosmotic agent. This step (without any additional osmotic agent in anormal nutrient medium) of incubation is repeated after 24 hourincubation in the dark at 25° C.

Alternatively, post-thawing of cells in a pellet upon thawing can bewashed quickly with a liquid medium containing an osmotic agent(sucrose, sorbitol or mannitol; concentration range: 1M to 2M). This isusually done by incubation of cell biomass for two to five minutes inliquid nutrient medium containing osmotic agent and centrifugation at100× g for one to three minutes. This step is repeated once more priorto the incubation of cell biomass for 30 minutes in liquid nutrientmedium containing osmotic agent and ethylene inhibitor or ethyleneaction inhibitor (silver thiosulfate at 5 μM to 20 μM).

New callus regrowth is usually visible after one week. When the new cellbiomass growth is increased, callus cells are removed from the filterpaper and transferred directly onto a normal solid nutrient medium.After sufficient growth (usually two to three weeks) has taken place, acell suspension in liquid medium is initiated from established callus.

Another embodiment of the invention is directed to plant cells whichhave been cryopreserved by the methods described above. Cells may be ofany genus or species disclosed or which the cryopreservation methods canbe applied. Cryopreserved cells may be maintained at temperaturesappropriate for cryo-storage. Preferably, cells are maintained in liquidnitrogen (about -196° C.), liquid argon, liquid helium or liquidhydrogen. These temperatures will be most appropriate for long termstorage of cells, and further, temperature variations can be minimized.Long term storage may be for months and preferably for many yearswithout significant loss of cell viability upon recovery. As theinvention also relates to efficient methods for recovery ofcryopreserved cells, relatively large portions of cell samples may belost without loss of the entire sample. Cells or plants can bepropagated from those cells that remain. Short term storage, storage forless than a few months, may also be desired wherein storage temperaturesof -150° C., -100° C. or even -50° C. may be used. Dry ice (carbondioxide) and commercial freezers may be used to maintain suchtemperatures.

Another embodiment of the invention is directed to plants and plantcells which have been revived by the cryopreservation recovery methodsdescribed above. These cells may also be of any of the genus or speciesdisclosed herein or a genus or species to which the methods ofcryo-recovery have been applied. Cells may be the original cells whichwere cryopreserved or cells which have proliferated from such cells. Aplant, as distinguished from a homogeneous cell culture, is a diversecollection of connected cells that possess interrelated functions.

Another embodiment of the invention is directed to methods and kits forthe transportation and thawing of cryopreserved cells. Cellscryopreserved by this method may be stored in a central repository forsubsequent retrieval. For increased safety against accidental loss, eachcell line frozen may be stored in a number of locations. Duringretrieval, a cryovial containing the cryopreserved cells may be shippedin a suitable container to the recipient. Suitable container are thosewhich can maintain cryopreservation temperature during shipment. Allcells can be shipped at temperatures sufficiently low for long termstorage with portable cryopreservation agents such as liquid nitrogen.Cells destined for immediate thawing may be shipped in dry ice to reducecost. A kit for the retrieval of cells from a repository may include avial of cryopreserved cells, sufficient media with the appropriateconcentrations of osmotic agents, vitrification solutions, andstabilizers for serial washes. Alternatively, in place or in addition tothe wash solution, the cells may be shipped with a plurality ofsemisolid growth media comprising a stabilizer and decreasing amounts ofosmotic and vitrification solutions. After thawing, the cells are eitherwashed and used immediately or they may be placed on the semisolid mediato gradually remove the vitrification and osmotic agents. The transportkit may further include reagents for an post thaw viability assay and areference DNA sample for comparison with DNA from the thawed cells todetermine genetic stability.

The following experiments are offered to illustrate embodiments of theinvention and should not be viewed as limiting the scope of theinvention.

EXAMPLES Example 1 Callus Initiation and Proliferation

Taxus needles were collected from wild and cultivated plants. Plantmaterial was washed in a diluted soap solution, rinsed extensively withdistilled water and surface sterilized in a chlorous solution (1%hypochlorite, pH 7) for 10 minutes. Under sterile conditions, thematerial was rinsed 3 times with sterile distilled water. Needles werecut in a 1% polyvinylpyrrolidone (PVP) solution with 100 mg/L ascorbicacid. Needles were placed with the cut end in semisolid medium E andincubated at 24° C.±1° C. in the dark. Cultures were monitored daily andthose with signs of contaminating microorganisms were discarded.Substantial callus formation was observed and the callus was separatedfrom the explant by 20 days and placed on the various callusproliferation media listed in Table 4. Calli of Taxus chinensis weretransferred to medium D (Table 4). This procedure resulted in callusinduction in over 90% of the explants. The same procedure wassuccessfully used to initiate cultures of T. brevifolia, T. canadensis,T. cuspidata, T. baccata, T. globosa, T. floridana, T. wallichiana, T.media and T. chinensis. Calli removed from the explant were cultivatedat 24° C. in the dark. Healthy parts of the callus were transferred tofresh medium every 10 days. The preferred growth and maintenance mediafor the invention are listed:

                                      TABLE 4                                     __________________________________________________________________________    Chemical Composition of Various Growth Medium                                 Chemical                                                                        Ingredient A B C D E F G H                                                  __________________________________________________________________________    Ammonium Nitrate                                                                       --  --  --  --  --  400 500 400                                        Ammonium Sulfate 134 -- 33.5 134 67 -- 134                                    Boric Acid 3 1.5 0.75 3 1.5 0.75 6.2 1.5                                      Calcium Chloride 113.2 -- 28.31 113.24 56.62 72.5 113.24 72.5                 (anhydrous)                                                                   Calcium Chloride -- 0 50 -- -- -- -- --                                       2H.sub.2 O                                                                    Calcium Nitrate -- 208.4 -- -- -- 386 -- 386                                  4H.sub.2 O                                                                    Cobalt Chloride 0.03 -- 0.006 0.025 0.0125 -- 0.025 --                        6H.sub.2 O                                                                    Cupric Sulfate 0.03 0.01 0.006 0.025 0.0125 0.25 0.025 0.25                   5H.sub.2 O                                                                    Na.sub.2 EDTA 2H.sub.2 O 37.3 -- 9.32 37.3 18.65 37.3 37.3 37.3                                                   Ferric Sulfate -- 2.5 -- -- -- --                                            -- --                                      Ferrous Sulfate 27.8 -- 6.95 27.8 13.9 27.8 27.8 27.8                         7H.sub.2 0                                                                    Magnesium Sulfate 122.1 366.2 30.5 122.09 61.04 180.7 122.09 180.7                                                (anhydrate)                               Manganese Sulfate 10 23.788 22.5 10 5 22.3 10 22.3                            H.sub.2 O                                                                     Molybdenum -- 0.001 -- -- -- -- -- --                                         Trioxide                                                                      Molybdic Acid 0.25 -- 0.062 0.25 0.125 0.25 0.25 0.25                         (sodium salt) 2H.sub.2 O                                                      Potassium Chloride -- 65 -- -- -- -- -- --                                    Potassium Iodide 0.75 0.75 0.175 0.75 0.375 -- 0.75 --                        Potassium Nitrate 2500 80 625 2500 1250 -- 2500 --                            Potassium -- -- 10 -- -- 170 -- 170                                           Phosphate                                                                     (monobasic)                                                                   Potassium Sulfate -- -- -- -- -- 990 -- 990                                   Sodium Phosphate 130.5 16.5 32.62 130.5 65.25 -- 130.5 --                     (monobasic                                                                    anhydrous)                                                                    Sodium Sulfate -- 200 -- -- -- -- -- --                                       Zinc Sulfate 7H.sub.2 O 2 3 0.5 2 1 8.6 2 8.6                                 Myo-Inositol 100 100 125 100 50 100 100 100                                   Nicotinic Acid 1 -- 0.75 1 0.5 1 1 1                                          Pyridoxine-HCl 1 -- 0.25 1 0.5 1 1 1                                          Thiamine-HCl 10 5 3.5 10 5 10 10 10                                           Glutamine 292.6 146.4 -- 292.8 292.8 1756.8 -- 292.8                          Tryptophan -- -- -- -- -- -- -- --                                            Phenylalanine -- 30 -- -- -- -- -- --                                         Lysine -- 20 -- -- -- -- -- --                                                Methionine -- -- -- -- -- -- -- --                                            Sodium Acetate  10 10 -- -- -- -- --                                          Sucrose 10000 50000 40000 10000 10000 10000 20000 10000                       N.sub.6 -Benzyladenine 0 2 2 0.002 0.002 --  --                               Ascorbic Acid 50 100 50 100 100 100 100 100                                   Picloram -- --  1.2 2.4 1.2 -- 1.2                                            Casein Hydrolysate -- -- 500 -- -- -- 1000 --                                 6- -- -- -- -- -- 0.02 -- --                                                  Dimethyltallylamino                                                           Purine                                                                        Kinetin -- -- -- -- -- -- -- 0.02                                             pH 5.6 5.6 5.6 5.6 5.6 5.6 5.6 5.6                                            β-Naphthaleneacetic 0.931 10 -- -- -- -- 1.862 --                        Acid                                                                        __________________________________________________________________________

Example 2 Suspension Initiation and Growth of Suspended Cells

One gram of callus material was aseptically inoculated into a 125 mlErlenmeyer flask containing 25 ml of liquid medium (Table 4). The flaskwas covered with a silicone foam cap and placed on a gyratory shaker at120 rpm at 24° C. in darkness. Suspension cultures were formed inapproximately 3-10 days. Medium exchanged was initiated by suctionfiltering the flask contents through a buchner funnel containing amiracloth filter and resuspending all the biomass in fresh medium. Oneto two grams of cells were transferred into a 125 ml flask containing 25ml of fresh medium weekly. Typical growth rates and cell densitiesachieved in suspension cultures of representative species are listed inTable 5.

                  TABLE 5                                                         ______________________________________                                        Growth Profile of Taxus Cells                                                              Dry Weight Fresh Weight                                                                           Dry Wt.                                                                              Fresh Wt.                               Species Doubling Time Doubling Time Density Density                         ______________________________________                                        T. brevifolia                                                                          2.0 days   3.5 days   20 g/L 400 g/L                                   T. baccata 2.0 days 6.0 days 15 g/L 220 g/L                                   T. chinensis 2.5 days 4.5 days 20 g/L 285 g/L                                 T. canadensis  8.5 days 13 g/L 260 g/L                                      ______________________________________                                    

The increase in biomass (fresh and dry weight) with time for T.chinensis line K-1 was plotted in FIG. 4. The maximal growth rate wasdetermined by measuring the slope at points of most rapid biomassincrease. Cell cultures of T. chinensis with a maximum doubling time of2.5 days, has a growth rate significantly higher than previouslyreported for Taxus species suspension cultures. Typical Taxus cultureshave doubling times of about 7-12 days.

Culturing cells at high density maximizes the productivity of the cellculture process. Whereas previous cultures of T. brevifolia has a celldensity of less than 1 gram dry weight per liter (Christian et al.,1991), suspension cultures T. chinensis can reach densities up to 8-20grams dry weight per liter after 18 days of growth. Cell viability wasdetermined by staining with a 0.05% fluorescein diacetate (FDA) inacetone (J. M. Widholm, Stain Technol 47:189-94, 1972) followed bycounting the number of green fluorescein cells upon excitation with bluelight in a fluorescence microscope. Cell viability was higher than 90%throughout the growth phase.

Example 3 Viability of Taxus Cells After Preculturing with Mannitol

Six to 7 day old suspension cultures of Taxus cells were harvested andresuspended into fresh growth medium containing 0.16M mannitol, 0.22Mmannitol, 0.33M mannitol or 0.44M mannitol.

After 3 days of incubation in growth medium with mannitol, cells werecold acclimated at 4° C. for 3 hours. Acclimated cells were harvestedand transferred to 4 ml cryovials containing a cold vitrifying solutionof 40/30 wt % ethylene glycol/sorbitol in media. The vials wereincubated at 4° C. for 3 minutes and frozen by immersion into liquidnitrogen. Vials were maintained in liquid nitrogen for at least 10minutes before use in the thawing experiments.

Vials of frozen cells were removed from liquid nitrogen storage andagitated at 40° C. until the contents are liquefied (1-2 minutes). Theliquefied cells were then washed 5 to 6 times with sterile mediacontaining 1 M sorbitol, 3 times with media containing 0.5 M sorbitolmedia, 3 times with 0.1 M sorbitol media, and 3 times with sorbitol freemedia. Washing was performed by resuspension of cells in wash media,centrifugation at 50× g for 2 minutes and aspiration of wash media fromthe cell pellet. Cell viability was determined immediately afterthawing. The summary of multiple experiments is listed below.

                  TABLE 6                                                         ______________________________________                                        Post Thaw Viability of Cells Pretreated with Mannitol                              Concentration                                                                            Viability       Regrowth                                      ______________________________________                                        0.16M       60%             vigorous                                            0.22M 30% slight                                                              0.33M 30% slight                                                              0.44M 20% slight                                                            ______________________________________                                    

Example 4 Viability of Taxus Cells After Preculturing With Sorbitol

Frozen Taxus cells were thawed and suspended into fresh growth mediumcontaining sorbitol at 0.15 M, 0.22 M, 0.33 M, 0.44 M and 0.80 Msorbitol. Cell viability was determined immediately after thawing. Asummary of the results from multiple trials are listed below:

                  TABLE 7                                                         ______________________________________                                        Post Thaw Viability of Cells Pretreated with Sorbitol                              Concentration                                                                            Viability       Regrowth                                      ______________________________________                                        0.15M       20%             none                                                0.22M 40% vigorous                                                            0.33M 30% vigorous                                                            0.44M 20% vigorous                                                            0.80M 20% slight                                                            ______________________________________                                    

Example 5 Viability of Taxus After Preculturing With Sucrose

Six to seven day old cell suspensions in growth medium were harvestedand the cell biomass resuspended in fresh growth medium containing 0.06M, 0.12 M, 0.15 M, 0.29 M and 0.58 M sucrose. Cells were cryopreserved,frozen, thawed and osmotically adjusted accordingly. Cell viability wasdetermined immediately after thawing. A summary of the results frommultiple experiments are listed in Table 8:

                  TABLE 8                                                         ______________________________________                                        Post Thaw Viability of Cells Pretreated with Sucrose                               Concentration                                                                            Viability       Regrowth                                      ______________________________________                                        0.06M       40%             slight                                              0.12M 40% slight                                                              0.15M 40% slight                                                              0.29M 30% slight                                                              0.58M <15%  slight                                                          ______________________________________                                    

Example 6 Effects of Osmotic Agents on the Survival of Taxus Cells

Various osmotic agents in growth medium were evaluated to determinetheir effects on the survival of Taxus species cells precultured withthe agents, after the preculture period and after thawing of thecryoprotected and frozen Taxus cell suspensions. Cells of three-day cellculture suspensions were precultured in growth medium containing variousosmotic agents prior to cryoprotection. Cryoprotected cells were frozenand stored in liquid nitrogen for a minimum of one hour. Viability testswere performed at the end of the preculture period (control) andimmediately after thawing of the cryoprotected and frozen cell.

Cell cultures pretreated with mannitol in growth medium exhibited thehighest percent viability upon thawing after cryoprotection and freezingas compared to viability observed using the other osmotic agents.

                  TABLE 9                                                         ______________________________________                                        Effects of Osmotic Agent on Post Thaw Viability                                                Concentration Survival                                         (Viability)                                                                 Osmotic Agent    Control Frozen                                               ______________________________________                                        Proline          <50%    <15%                                                   Trehalose 50-95% <15%                                                         Sucrose 50-95% 20-50%                                                         Sorbitol 50-95% 30-70%                                                        Mannitol 50-95% 40-80%                                                      ______________________________________                                    

Example 7 Effect Osmotic Agents and Cryoprotectants on Taxus Viability

Cells of Taxus suspension cultures (KS1A) were harvested and preculturedwith various osmotic agents in the medium. Osmotic agents tested includetrehalose, proline, sorbitol (0.15 M-0.8 M), sucrose (2-20%) andmannitol (0.16 M). Viability was evaluated for each cell suspension atthe end of the preculture period and immediately after thawing. Regrowthwas evaluated after post-thaw osmotic adjustment. The vitrificationsolutions used were ethylene glycol/sorbitol/pectin and ethyleneglycol/sorbitol at 40/30 weight percent in culture medium. The resultsare summarized in Table 10. The highest percent viability and mostrigorous regrowth were observed when mannitol was used for preculturingand ethylene glycol/sorbitol was used as the cryoprotectants in thevitrification solution.

                  TABLE 10                                                        ______________________________________                                        Effects of Osmotic Agents and Cryoprotectants of Post Thaw Viability            Osmotic         Post-Thaw   Recovery                                        Agent    Viability                                                                              Cryoprotectants                                                                           Viability                                                                            Growth                                   ______________________________________                                        Trehalose                                                                              50-95%   Ethylene glycol/                                                                          <15%   none                                       and Proline  Sorbitol/pectin                                                  Sorbitol 50-95% Ethylene glycol/ 30-70% slight to                             0.15M-  Sorbitol  vigorous                                                    0.8M                                                                          Sucrose 50-95% Ethylene glycol/ <10-40% none to                                2%-10% Sorbitol  vigorous                                                    Mannitol 75-95% Ethylene glycol/ 40-80% slight to                               Sorbitol  vigorous                                                        ______________________________________                                    

Example 8 The Effect of Preculture Length on Survival

Taxus cells were harvested from cell culture and the biomass resuspendedin growth medium containing mannitol at a concentration of 3% for oneday or three-days at room temperature. Loaded cells were incubated at 4°C. for 3 hours and transferred to 4 ml cryovials containing coldvitrifying solution which comprising 40/30 weight percent ethyleneglycol/sorbitol in culture medium. The vials were incubated at 4° C. forthree minutes and frozen by liquid nitrogen immersion. Cells containedin the vials were maintained in liquid nitrogen for at least 10 minutes.

Cryopreserved cells were thawed and their viability was determined byFDA and trypan blue staining procedures. Cells precultured with mannitolfor three days exhibited significantly higher post-thaw availabilitythan cells which were not precultured in medium containing mannitol.

                  TABLE 11                                                        ______________________________________                                        Effects of Preculture Time on Viability                                         Days of           Survival                                                                              3%                                                  Preculture Control Mannitol                                                 ______________________________________                                        1               5-10%   50%                                                     3 5-15% 40-80%                                                              ______________________________________                                    

Example 9 Effect of Ethylene Glycol/Sorbitol on Thawed Taxus CellViability

Six to seven day cell suspensions of Taxus species cell line KS1A werepretreated with 3% mannitol for three days at room temperature. Loadedcells were acclimated to the cold by incubating the flasks at 4° C. for3 hours. Cold acclimated cells were transferred to 4 ml cryovials andcold vitrification solution was added to each and mixed. Aftervitrification at 4° C. for three minutes, cells were frozen by liquidnitrogen immersion. Vials were maintained in liquid nitrogen for atlease 10 minutes.

Cryopreserved cells were thawed by transferring vials from liquidnitrogen and agitated in a 40° C. water bath for 1-2 minutes. Post-thawviability was determined by FDA staining assay.

Ten trials evaluating cell suspensions of Taxus species cell line KS1Awere performed with different concentrations of ethyleneglycol/sorbitol. The results are summarized in Table 12. Cellsuspensions frozen in the vitrification solution containing ethyleneglycol/sorbitol at a concentration of 40/30 wt %, exhibited the highestpost-thaw percent viability as well as the most vigorous regrowth ascompared to cells vitrified using other concentrations of ethyleneglycol/sorbitol.

                  TABLE 12                                                        ______________________________________                                        Effects of Vitrification Solution of Recovery                                      Ethylene Glycol/                                                                          Post-Thaw                                                      Sorbitol Viability Regrowth                                                 ______________________________________                                        50%/30%      20%             slight                                             45%/35% 25% slight                                                            40%/40% 25% slight                                                            40%/30% 60% vigorous                                                          38%/32% 40% vigorous                                                          36%/34% 35% moderate                                                          35%/35% 35% moderate                                                          30%/40% 40% slight                                                            30%/40% 40% slight                                                            20%/50% 25% none                                                            ______________________________________                                    

Example 10 Effects of Cryoprotectants of Taxus Cells Survival After-196° C. Storage

Cultured Taxus cells were harvested and resuspended in fresh growthmedium containing 3% mannitol for 3 days. Cells were cold acclimated for3 hours and transferred to cryovials containing vitrification solution.Cell suspensions were frozen by liquid nitrogen immersion. Liquidnitrogen frozen cells were thawed by agitating the cryovials in a 40° C.water bath for 1-2 minutes. Cells frozen with ethylene glycol as acryoprotectant has the highest viability.

                  TABLE 13                                                        ______________________________________                                        Effects of Cryoprotectants on Viability                                            Cryoprotectant                                                                              Concentration Viability                                    ______________________________________                                        DMSO           5%-30%        ≦15%                                        Propylene Glycol 15 % 0                                                       Glycerol 20%-30% 0                                                            PEG-8000 10% 0                                                                Ethylene Glycol 20%-50% 25%-80%                                             ______________________________________                                    

Example 11 Viability of Taxus Cells as a Function of Biomass toVitrifying Solution

Six to seven day old cell suspension of taxus species cell line KS1Awere harvested and resuspended in fresh medium containing 3% mannitoland incubated for three days at room temperature. Following coldacclimation at 4° C. for 3 hours, cells were transferred to 4 mlcryovials containing 40/30 wt % ethylene glycol/sorbitol in culturemedium. Vials were incubated at 4° C. for 3 minutes and frozen by liquidnitrogen immersion. Vials were maintained in liquid nitrogen for atleast 10 minutes before thawing.

The highest percent viability was observed when the cellbiomass/vitrifying solution quantity was 167 mg/ml. Acceptable viabilitywas also when the cell biomass/vitrifying solution ratio was 143, 200and 250 mg/ml.

                  TABLE 14                                                        ______________________________________                                        Effects of Cell Biomass on Viability                                                  Cell Biomass/                                                           Vitrifying Solution Viability                                               ______________________________________                                          143 mg/ml      60%                                                              167 mg/ml 80%                                                                 200 mg/ml 45%                                                                 250 mg/ml 45%                                                                 300 mg/ml ≦10%                                                         400 mg/ml ≦10%                                                         500 mg/ml ≦5%                                                        1,000 mg/ml ≦5%                                                      ______________________________________                                    

Example 12 Effects of Different Method Steps on Taxus Cell Viability

Different method steps were evaluated to determine the steps which wouldresult in the highest percent post-thaw viability. Cells werecryopreserved with and without cryoprotectants, with and without osmoticpretreatments, with and without cold treatment, and with and withoutvitrification.

In the first trial, six to seven day old Taxus cells cultures werefrozen with and without cryoprotectants. In the second trial, cells werefrozen with and without a 40/30, weight percent, ethyleneglycol/sorbitol vitrification solution treatment. In the third trial,cells were vitrified and frozen with and without a pretreatmentcomprising a three day incubation in 3% mannitol growth media. In thefourth trial, cells were pretreated and vitrified and frozen with orwithout cold acclimation.

For each trial, viability tests were performed immediately afterthawing. Cells precultured with growth medium containing 3% mannitol for3 days at room temperature, followed by a 2-4 hour cold treatment priorto cryoprotection, exhibited the highest percent viability. Suitableviability was also observed in cells precultured for 3 days in mediumcontaining 3% mannitol and subjected to cryoprotection without previouscold treatment, and in cells preculture in growth medium for 3 days andprecultured in media containing mannitol for 24 hours followed by a 2 to4 hour cold treatment prior to cryoprotection.

                  TABLE 15                                                        ______________________________________                                        Viability of Cells Recovered from Liquid Nitrogen                                  Treatment     Viability     Regrowth                                     ______________________________________                                        Cells in medium                                                                              0             none                                               Direct plunging into                                                          liquid nitrogen                                                               Cells in medium ≦10 none                                               cryoprotection                                                                liquid nitrogen                                                               Cells in medium 40-60 slight                                                  precultured 3 days                                                            in 3% mannitol                                                                cryoprotection                                                                liquid nitrogen                                                               Cells in medium 40-80 vigorous                                                Precultured 3 days                                                            in 3% mannitol                                                                2-4 hour cold                                                                 treatment                                                                     cryoprotection                                                                liquid nitrogen                                                               Cells in growth 40-60 vigorous                                                medium for 3 days                                                             preculture in osmotic                                                         media for 24 hours                                                            3% mannitol                                                                   2-4 hour cold                                                                 treatment                                                                     cryoprotection                                                                liquid nitrogen                                                             ______________________________________                                    

Cells were again tested for viability tests using the indicated steps,performed according to the methods described herein.

                  TABLE 16                                                        ______________________________________                                        Viability of Cells Recovered from Liquid Nitrogen                                   Treatment  Viability       Regrowth                                     ______________________________________                                        Freeze Dried 20%             none                                               Liquid nitrogen                                                               Freeze Dried 30-60% moderate                                                  Vitrification                                                                 Liquid nitrogen                                                               Freeze Dried 20-40% slight                                                    Preculture                                                                    in sorbitol                                                                   Liquid nitrogen                                                               Freeze Dried 30-60% moderate                                                  Preculture                                                                    in sorbitol                                                                   Vitrification                                                                 Liquid nitrogen                                                               Freeze Dried 20-40% slight                                                    Loading                                                                       Vitrification                                                                 Liquid nitrogen                                                               Freeze Dried 30-50% good                                                      Loading                                                                       Vitrification                                                                 Liquid nitrogen                                                               Preculture 40-60% good                                                        in sorbitol                                                                   Freeze Dried                                                                  Vitrification                                                                 Liquid nitrogen                                                               Preculture 40-60% good                                                        in mannitol                                                                   Freeze Dried                                                                  Vitrification                                                                 Liquid nitrogen                                                               Preculture 40-60 % good                                                       in sucrose                                                                    Freeze Dried                                                                  Vitrification                                                                 Liquid nitrogen                                                             ______________________________________                                    

Example 13 Cell Viability and Growth Before and After Cryopreservation

The following Taxus species cell lines were evaluated to determine cellviability and regrowth after cryopreservation: KS1A; KEIR; 647; 1224;12-6;12-14; and 12-20.

Six to seven day old cell suspensions of each cell line were harvestedand the biomass resuspended in fresh growth medium containing 3%mannitol. Cells were incubated for 3 days at room temperature andthereafter the loaded cells suspensions were incubated at 4° C. for from3 hours. Cold acclimated cells were transferred to 4 ml cryovialscontaining a cold vitrification solution of ethylene glycol/sorbitol40/30 wt %. The vitrification solution and cells were gently mixed andthe vials were incubated at 40° C. for 3 minutes. Thereafter, the cellsuspensions were frozen by liquid nitrogen immersion for at least 10minutes.

After freezing, the cells were thawed by transferring the frozen vialsfrom liquid nitrogen to a 40° C. water bath for 1-2 minutes. Post-thawcell viability was determined by FDA staining assay. Cells were washed5-6 times with cold sterile 1M sorbitol media and resuspended in fresh 1M in medium.

The cell suspensions free from toxic cryoprotectants were then eachseparately filtered using a buchner funnel and Whatmann 541 filter paperunder sterile conditions. For each cell suspension, the filter withcells was layered on semisolid growth medium containing 0.5M sorbitoland equilibrated for 30 minutes at room temperature. Paper containingcells was transferred to solid growth medium containing 0.1M sorbitoland incubated for 24 hours. The paper with cells was transferred tosemisolid growth medium without sorbitol and incubated for 24 hours atroom temperature. The filter containing cells was then again transferredto fresh semisolid growth medium without sorbitol and incubated at roomtemperature for an additional 24 hours. Callus cell growth on thesemisolid nutrient media was evident at about 2 to 3 weeks. Thereafter,cell suspensions in liquid growth medium were initiated from the callus.

As can be seen from Table 17 set forth below, all of the cell linesevaluated exhibited acceptable post-thaw viability and recovery growth.

                  TABLE 17                                                        ______________________________________                                        Effects of Preculture Conditions on Viability                                   Cell    Preculture       Cryo-   Post-Thaw                                                                            Recovery                              Line Condition Viability Protectants Viability Growth                       ______________________________________                                        KS1A  3% mannitol                                                                             ≧95%                                                                            ethylene                                                                              40-80% vigorous                                 3 days  glycol/                                                                 sorbitol                                                                      40/30 wt %                                                                 KEIR 3% mannitol ≧95% ethylene 30-60% slight                            3 days  glycol/                                                                 sorbitol                                                                      40/30 wt %                                                                 647 3% mannitol ≧95% ethylene 30-60% slight                             3 days  glycol/                                                                 sorbitol                                                                      40/30 wt %                                                                 1224 3% mannitol ≧95% ethylene 40-60% vigorous                          3 days  glycol/                                                                 sorbitol                                                                      40/30 wt %                                                                 12-6 3% mannitol ≧95% ethylene 40% vigorous                             3 days  glycol/                                                                 sorbitol                                                                      40/30 wt %                                                                 12-14 3% mannitol ≧95% ethylene 30% vigorous                            3 days  glycol/                                                                 sorbitol                                                                      40/30 wt %                                                                 1220 3% mannitol ≧95% ethylene 35% vigorous                             3 days  glycol/                                                                 sorbitol                                                                      40/30 wt %                                                               ______________________________________                                    

Example 14 Growth and Product Formation of Taxus Cells UponCryopreservation

Six to seven day cell suspensions of Taxus cell line KS1A werecryopreserved and thawed. The cells were precultured with 3% mannitol ingrowth medium for 3 days and 40/30 wt % ethylene glycol/sorbitol wasused as a cryoprotectants. Growth doubling time and product formationwere evaluated before and after freezing and thawing. Product yield wasmonitored after 5 days of growth in suspension. Nuclear DNA content inthe cells was monitored by flow cytometry and found to be about 22.7pg/nuclei before and 22.9 pg/nuclei after cryopreservation.Cryopreservation did not affect product production.

                  TABLE 18                                                        ______________________________________                                        Growth and Product Formation of Taxus before and after LN.sub.2                 Preculture                                                                              Cryo-     (Doubling                                                                            Product Formation (mg/L)                         Treatments                                                                            Protectant                                                                              Time)    Taxol Baccatin                                                                             10-DAB                                ______________________________________                                        3% mannitol                                                                           ethylene  7 (5-7)  0.2   1.1    0.4                                      glycol/                                                                       sorbitol/                                                                     (40/30 wt %)                                                                  -- 7 (5-7) 0.2 1.1 0.4                                                     ______________________________________                                    

Example 15 Growth and Product Formation in Taxus Cells AfterCryopreservation

Taxus species cell lines were cryopreserved and subsequently thawed andsubjected to post-thaw osmotic adjustment. Growth and product formationwere determined after the establishment of cell suspensions in liquidculture. Growth reflects the average doubling time of cell suspensionsin days after they were established in growth medium. Product formationwas determined after 14 days of growth in suspension. Results are listedin Table 19.

                  TABLE 19                                                        ______________________________________                                        Taxol Production of Cells Recovered from Cryopreservation                                   (Average    Production                                            Cell doubling time (mg/L - 14 days)                                         Line      in days)    Taxol   Baccatin                                                                              10-DAB                                  ______________________________________                                        Keir      4           10.5    10.6    3.4                                       KS1A 5.5 22.6 7.4 2.3                                                         1224 5.5 10.8 73 13                                                           SS3-184 7 --  -- --                                                           SS12-6 5.8 25 51 6.7                                                          SS12-19 6 9.2 31 4.7                                                          SS12-20 5.5 10 24 4.4                                                         SS12-79 5 2.4 9.6 1.9                                                         SS12-99 8.5 -- -- --                                                          SS12-103 6 19.3 68.5 14.3                                                     647 5.5 -- -- --                                                            ______________________________________                                    

FIG. 5 illustrate chromatograms of the extracellular fraction at day 20from Taxus species cell line 1224 where (A) represents the control cellsuspension Which was not cryopreserved and (B) represents the cellsuspension regenerated after cryopreservation, freezing, storage for sixmonths, and thawing and post-thaw osmotic adjustment. Peak 1 is10-deacetylbaccatin; peak 2 is 9-dihydrobaccation III; peak 3 isbaccatin III; peak 4 is 9-dihydro-13-acetylbaccatin III; peak 5 istaxol; peak 6 is 2-benzoyl-2-deacetylbaccatin and peak 7 is2-benzoyl-2-deacetyl-1-hydrozybaccatin I.

FIG. 6 illustrate chromatograms of the extracellular fraction at day 20from Taxus species cell line 203 where (A) represents the control cellsuspension Which was not cryopreserved and (B) represents the cellsuspension regenerated after cryopreservation, freezing, storage forthree months, and thawing and post-thaw osmotic adjustment. Peak 1 is10-deacetylbaccatin; peak 2 is 9-dihydrobaccation III; peak 3 isbaccatin III; peak 4 is 9-dihydro-13-acetylbaccatin m; peak 5 is taxoland peak 6 is 2-benzoyl-2-deacetylbaccatin. As can be seen from FIGS. 5and 6, the product formation profile is substantially the same in thecontrol cell suspension and the regenerated cell suspension.

Example 16 Genetic Stability of Cryopreserved and Non-CryopreservedCells

Cell lines were established from a single Taxus chinensis var. mairertree. One of these established cell lines was cultured, cryopreservedfor one year, and thawed. Genetic analysis was performed on cells fromthe original tree and on the cryopreserved cells to determine ifcryopreservation have affected genetic stability of the cells. Briefly10 μg of total DNA from each cell line was and treated with a four foldover digestion of restriction endonuclease and size fractionated byagarose gel electrophoresis. The size fractionated DNA was transferredto a nitrocellulose solid support and hybridized to a radio labelednucleic acid probe, Jeffrey's 33.6 minisatellite probe. Thishypervariable region probe shows different banding patterns from the DNAof 4 separate trees in lanes 1-4 of FIG. 7. In contrast, the initialisolate (lane A), cells cultured for 1 year (lane B), and cellscryopreserved for 1 year (lane C) was identical genetically from cellsisolated from the same tree one year later (lanes D and E).Cryopreservation did not result in any mutation detectable by thisanalysis.

Example 17 Stability of Cryopreserved Taxus Cells

To determine if the length of cryopreservation has any effect on geneticstability, Taxus cell lines 1224 was frozen for one hour to 6 months andanalyzed for their genetic stability. DNA was extracted from viablecultures grown thawed and re-established from these cryopreserved cells.A 3.1 Kb polymorphic region of the genome containing nuclear ribosomalcoding and non-coding DNA was amplified by polymerase chain reaction anddigested with endonuclease DpnII. The digested DNA was sorted by sizeusing gel electrophoresis and visualize after ethidium bromide staining.The results of the analysis is shown in FIG. 8. The original cell lineand a noncryopreserved cell line were analyzed in lanes C and Drespectively. Two unrelated cell lines established from unrelated treesshow a different digestion pattern. In contrast, no genetic mutation wasdetected in cells cryopreserved for one hour (lane E), one day (lane F),one week (lane G), one month (lane H) or 6 months (lane I and J). Thebanding pattern of these cryopreserved and non-cryopreserved cells wereall identical (lanes C to J).

Example 18 Cryopreservation of Tomato Cell Lines

For successful cryopreservation of tomato cell lines, loading andvitrification solutions were added gradually in a stepwise fashion toreduce osmotic shock. Loading and vitrification solutions comprised of30% (w/v) glycerol, 15% (w/v) ethylene glycol and 15% (w/v)dimethylsulfoxide. Without gradual addition, cells did not grow rapidlyafter the post-thawing recovery period. The effect of post-thawviability and recovery regrowth was examined and the results are shownin Table 20.

                  TABLE 20                                                        ______________________________________                                        Effects of Preculturing Conditions on Viability                                 and Recovery Regrowth of a Tomato Cell Line                                             Preculturing Duration (Percent Viability                          Osmotic Agent - Conc.                                                                     1 Day    2 Days   3 Days  6 Days                                  ______________________________________                                        Sucrose -                                                                            0.06 M   5 (-)    10 (-) 15 (+)  15 (+)                                   0.1 M 20 (+) 30 (++) 35 (++) 40 (++)                                          0.3 M 35 (++) 35 (++) 50 (+++) 60 (+++)                                       0.5 M 45 (+++) 60 (+++) 65 (+++) 30 (++)                                      0.8 M 50 (+++) 30 (+++) 10 (-) 10 (-)                                        Sorbitol - 0.06 M 10 (-) 15 (+) 20 (+) 30 (+)                                  0.1 M 15 (+) 20 (++) 40 (++) 60 (+++)                                         0.3 M 30 (++) 40 (++) 70 (+++) 50 (+++)                                       0.5 M 20 (++) 40 (++) 35 (++) 10 (+)                                          0.8 M 10 (-) 30 (+++) 20 (+) 5 (-)                                           Mannitol - 0.06 M 5 (-) 5 (-) 10 (-) 10 (-)                                    0.1 M 15 (+) 30 (++) 35 (++) 40 (++)                                          0.3 M 40 (++) 45 (++) 50 (+++) 20 (+)                                         0.5 M 30 (++) 40 (++) 35 (++) 15 (+)                                          0.8 M 20 (+) 35 (++) 15 (+) 5 (-)                                          ______________________________________                                         (-) = no recovery regrowth of callus                                          (+) = slight recovery regrowth of callus                                      (++) = moderate recovery regrowth of callus                                   (+++) = vigorous recovery regrowth of callus                             

The highest viability (survival of cells) was obtained when cells wereprecultured for three days in liquid nutrient media containing 0.5 Msucrose or 0.3 M sorbitol, 65% and 70% respectively. These preculturingconditions also supported vigorous growth of cells upon post-thawing ofcell suspensions. Successful cryopreservation also depended on thepreculture period and the concentration of the osmotic agent. Prolongingthe preculture period to ten days and increasing the concentrations ofosmotic agents to greater than 0.8M did not increase viability of thecells. Without preculture, cells did not survive LN₂ temperatures eitherwith one step or with stepwise loading and vitrification solutionaddition methods.

Viability and regrowth recovery was further enhanced by exposingprecultured cells to a heat-shock treatment up to four hours, and by theaddition of divalent cations (CaCl₂, MgCl₂). This approach has also beensuccessfully used in the cryopreservation of cell lines derived fromspecies of Lupinus, Sophora, Conospernum, Nicotiana and Solanum as wellas the recalcitrant cell lines derived from various Taxus species suchas Taxus chinensis. In many instances, regrowth of cell lines of theseplant species was macroscopically visible 3 to 4 days after plating ascompared to 6 to 10 days with one step loading and vitrificationmethods. Post-thaw wash of cell biomass with liquid medium containingethylene action inhibitors or ethylene biosynthesis inhibitors furtherimproved the quality of recovered cell lines. Viability rates of 90%were consistently achieved and faster growth rates were observed aftercell suspensions were established from these lines.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All U.S. patents cited herein arehereby specifically incorporated by reference in their entirety. Thespecification and examples should be considered exemplary only with thetrue scope and spirit of the invention indicated by the followingclaims.

I claim:
 1. A method for cryopreserving a plant cell comprising thesteps of: preculturing the plant cell with a divalent cation at greaterthan about 5 mM and an osmotic agent for a period of time effective tostabilize cell membranes; and in any order, loading the plant cell witha cryoprotecting agent; and vitrifying the plant cell with acryoprotection solution; and freezing the plant cell at acryopreservation temperature.
 2. The plant cell of claim 1 which is aspecies of Taxus, Solanum, Legume, Lycopersicum, Abies, Cypressus,Ginkgo, Juniperus, Picea, Pinus, Pseudotsuga, Sequoia, Tsuga, Zamia,Avena, Cocos, Dioscorea, Hordeum, Musa, Oryza, Saccharum, Sorghum,Triticum, Zea, Achyrocline, Atropa, Brassica, Berberis, Capsicum,Catharanthus, Conospermum, Datura, Daucus, Digitalis, Echinacea,Eschscholtzia, Glycine, Gossypium, Hyoscyamus, Malus, Medicago, Panax,Pisum, Rauvolfia, Ruta, Trichosanthes or Nicotiana.
 3. The plant cell ofclaim 2 wherein the Taxus species is T. baccata, T. brevifolia, T.canadensis, T. chinensis, T. cuspidata, T. floridana, T. globosa, T.media, T. nucifera or T. wallichiana.
 4. The method of claim 1 whereinthe plant cell is obtained from new growth needles, bark, leaves, stem,root, rhizome, callus cells, protoplasts, cell suspensions, meristems,seeds or embryos.
 5. The method of claim 1 wherein preculturing involvesculturing said plant cell in medium containing the osmotic agent and thedivalent cation for between about 1 day and about 6 days.
 6. The methodof claim 1 wherein the osmotic agent is sucrose, sorbitol or mannitol ata concentration of between about 0.06 M and about 0.8 M.
 7. The methodof claim 1 wherein the divalent cation is CaCl₂, MgCl₂ or MnCl₂.
 8. Themethod of claim 1 wherein the divalent cation is at a concentration offrom about 5 mM to about 20 mM.
 9. The method of claim 1 wherein loadingcomprises incubating said plant cell in the cryoprotection solutioncomprising between about 0.5% to about 30%, by weight, of thecryoprotecting agent.
 10. The method of claim 9 wherein thecryoprotecting agent is selected from the group consisting of DMSO,propylene glycol, glycerol, polyethylene glycol, ethylene glycol,butanediol, formamide, propanediol, sorbitol, mannitol and mixturesthereof.
 11. The method of claim 1 wherein the cryoprotecting agent isselected from the group consisting of DMSO, propylene glycol, glycerol,polyethylene glycol, ethylene glycol, butanediol, formamide,propanediol, sorbitol, mannitol and mixtures thereof.
 12. The method ofclaim 1 wherein the osmotic agent and the cryoprotecting agent are thesame.
 13. The method of claim 1 wherein loading and vitrifying areconducted simultaneously.
 14. The method of claim 1 wherein loading orvitrifying is performed in a single step or in a plurality of steps. 15.The method of claim 14 wherein the plurality of steps comprises addingthe cryoprotecting agent to the plant cell five times at one minuteintervals.
 16. The method of claim 1 wherein the cryopreservativetemperature is less than about -70° C.
 17. The method of claim 1 furthercomprising the step of including a stabilizer during preculturing,loading or vitrifying.
 18. The method of claim 17 wherein the stabilizeris a divalent cation, an oxygen radical scavenger, an antioxidant, anethylene inhibitor, a compound that intercalates into the lipid bilayerof the cell or a heat-shock protein.
 19. The method of claim 18 whereinthe ethylene inhibitor is an ethylene biosynthesis inhibitor or anethylene action inhibitor.
 20. The method of claim 1 further comprisingthe step of storing the cryopreserved cell at said cryopreservationtemperature for a period of time of greater than one month.
 21. Themethod of claim 20 wherein the period of time is greater than one year.22. A method for recovering cryopreserved plant cells comprising thesteps of:a) cryopreserving plant cells according to the method of claim1; b) thawing the cryopreserved plant cells to a temperature abovefreezing; c) incubating the thawed plant cells in a growth mediumcontaining a stabilizer; d) removing the cryoprotecting agent; and e)recovering viable plant cells.
 23. The method of claim 22 wherein thestabilizer is a divalent cation, an oxygen radical scavenger, anantioxidant, an ethylene inhibitor, a compound that intercalates intothe lipid bilayer of the cell or a combination thereof.
 24. The methodof claim 23 wherein the growth medium further contains a cyroprotectant.25. The method of claim 1 wherein the cryoprotection solution contains adivalent cation, which may be the same or different from the divalentcation used in preculturing.
 26. The method of claim 1 whereinpreculturing is performed simultaneously with loading and the osmoticagent and the cryoprotecting agent are the same.
 27. The method of claim1 wherein preculturing is performed simultaneously with vitrifying andthe cryoprotection solution contains the osmotic agent.
 28. A method forcryopreserving a plant cell comprising the steps of:preculturing theplant cell with a divalent cation at greater than about 5 mM and anosmotic agent for a period of time effective to stabilize cellmembranes; loading the plant cell with a cryoprotecting agent;vitrifying the plant cell with a cryoprotection solution; and freezingthe vitrified plant cell at a cryopreservation temperature.